Generation Of Induced Pluripotent Stem Cells From Amniotic Fluid Cells Of A Fetus With Hb Bart’s Syndrome

α-thalassemia is a group of hereditary hemolytic anemia caused by the lack or deficiency of α-globin chain synthesis due to the absence or mutation of α-globin gene.It is one of the most common human monogenic disease.Clinically,α-thalassemia is divided into silent carrier state,α-thalassemia trait,Hb H disease and the hemoglobin Bart’s hydrops foetus syndrome.Hemoglobin Bart’s hydrops foetus syndrome is also known as homozygous α-thalassemia(--SEA/--SEA),mainly results from the loss of all four α-globingenes(--/--).Because of the complete lack of α-globin chain synthesis,resulting in severe fetal anemia.If not intrauterine therapy,the fetus died more in the third trimester or died a few hours after birth.Hemoglobin Bart’s hydrops foetus syndrome is an important global health problem.Induced pluripotent stem cells(iPSCs)are pluripotent stem cells that can be produced by simultaneously expressing several transcription factors expressed in high levels of embryonic stem cells(ESC)in already differentiated somatic cells.The stem cells are similar to embryonic stem cells and have the unique ability to self-renew indefinitely while maintaining the potential to give rise to all cell types in the human body.Thus,the generation of induced pluripotent stem cells can avoid the ethical problems resulting from the use of human embryonic stem cells,and establish individualized disease-specific iPSCs to treat hereditary and degenerative diseases that avoid the risk of immune rejection.Objective To generate integration-free induced pluripotent stem cell lines from human amniotic fluid cells with Hemoglobin Bart’s hydrops foetus syndrome(--SEA/--SEA),in order to provide a model for gene repair and individualized intrauterine therapy.Methods 1.After obtaining informed consent,10 ml of amniotic fluid was drawn at 16 weeks of gestation by ultrasound-guided amniocentesis from a pregnant woman in whom the fetus was diagnosed as homozygous α-thalassemia(--SEA/--SEA)in Guangzhou Women and Children Medical Center for this study.2.The generation of the induced pluripotent stem cell line from human amniotic fluid cells with homozygous α-thalassemia(--SEA/--SEA),was carried out using nonintegrative Sendai virus vector containing the reprogramming factors(OCT3/4,SOX2,c-Myc,KLF4).Labeled h-AF-Se V-iPSCs.3.The pluripotency identification of h-AF-Se V-iPSCs was carried out by alkaline phosphatase staining and immunofluorescence staining.The ability of h-AF-Se ViPSCs to differentiate into three germ layers in vitro and in vivo was identified by embryoid body formation assay and teratoma formation assay.4.G-banding karyotype analysis were perform in h-AF-Se V-iPSCs at passages 10 and 20 to evaluate the stability of genetic material.The DNA of h-AF-Se V-iPSCs and its parental amniotic fluid cells were extracted.SEA types of α-thalassemia genotypes were identified to exclude cell contamination.5.6 × 104 amniotic fluid cells transduced by Sendai virus were inoculated on feeder cells.The time of cloning formation was observeand and the number of ES-like clones(alkaline phosphatase staining positive)was calculated.Results 1.The integration-free iPSCs from human amniotic fluid cells with homozygous α-thalassemia were successfully derived.2.h-AF-Se V-iPSCs exhibited strong alkaline phosphatase(AP)activity.Immuno fluorescence staining showed that h-AF-Se V-iPSCs expressed embryonic stem cell specific proteins Oct4,Sox2,SSEA-4 and Tra-1-81.3.h-AF-Se V-iPSCs were injected into the inguinal grooves of 7-week-old male severe combined immunodeficiency(SCID)mouse.The mouse were sacrificed 10 weeks after injection for teratoma examination.The teratomas were fixed,sliced,stained with hematoxylin and eosin,and immunohistochemical staining for further analysis.HE staining and immunohistochemical staining showed that teratomas contained endoderm,mesoderm and ectoderm tissues.h-AF-Se V-iPSCs can be formed into cystic embryoid bodies after suspension culture,and can be differentiated into three germ layers after adherent culture.4.G-banding Karyotype analysis of h-AF-Se V-iPSCs at passages 10 and 20 showed normal karyotypes(46,XY).h-AF-Se V-iPSCs and its parental amniotic fluid cells were detected by Gap-PCR,gel electrophoresis of PCR product showed homozygous α-thalassemia deletion.5.Four days after transdu(--SEA/--SEA)ction,the typical human ES-like colonies appeared.Seven days after transduction,the cells were plated on irradiated mouse embryonic fibroblast(MEFs)feeder layers.Nine days after transduction,human amniotic fluid cells began to aggregate,cells reprogrammed by Sendai viruses exhibited typical iPSCs morphology that showed compact cells within colonies of distinct margin.There were 30 ES-like clones were found,and the colones exhibited strong alkaline phosphatase(AP)activity.The efficiency for transduction was approximately 0.05%.Conclusion 1.Human amniotic fluid cells with homozygous α-thalassemia can be successfully induced into integration-free iPSCs in vitro.2.h-AF-Se V-iPSCs have the ability of self-renew and retain the potential to be differentiated into all cell types of the human body in vivo and in vitro.The iPSCs can maintain the stability of genetic material even culture in vitro long-term.Therefore,h-AF-Se V-iPSCs can be used as an ideal cell model for the treatment of homozygous α-thalassemia(--SEA/--SEA).3.Human amniotic fluid cells can be used as an ideal source for iPSCs.