Construction On A Cell Line Of Induced Pluripotent Stem Cells From A Patient Carrying Complex Heterozygous Mutations Of ABCA3 In NRDS

Objectives:To construct iPSCs a cell line from peripheral blood mononuclear cells（PBMCs）of patient with neonatal respiratory distress syndrome（NRDS）carrying complex heterozygous mutations of ATP binding box transporter A3（ABCA3）;To lay the foundation for clinical cell therapy for NRDS carrying complex heterozygous mutationsof ABC A3;And provide a disease cell model for studying the pathogenesis of NRDS with gene mutations of ABC A3.Methods:Pedigree investigation combined with gene exon sequencing was confirmed a case of NRDS carrying ABCA3 gene mutation.PBMCs were isolated from patient’s peripheral blood and cultured in vitro,then reprogramming factors were introduced into PBMCs through the non integrated Sendai virus vector,and iPSCs were obtained.IPSCs Continuously were subcultured with stem cell culture technology until the 40th generation.To identify the pluripotency of iPSCs,the qualitative expression of pluripotency markers were detected with immunofluorescence staining,the quantitative expression of pluripotency markers were tested with flow cytometry,and Gene expression of stem factor was determinedwith quantitative polymerase chain reaction（qPCR）.In order to identify the differentiation potential of iPSCs,markers in three embryonic layers including endoderm,mesoderm and ectoderm were detectd with immunofluorescence staining.The identity of cell line of iPSCs was confirmed with methods such as full exon gene sequencing,chromosome karyotype,and short tandem repeat（STR）site analysis.Sendai virus residues and viruses and microorganisms such as Mycoplasma were detected with Reverse transcription polymerase（RT-PCR）,so as to storage and recovery of iPSCs.Vitality of storing and resuscitating iPSCs was tested with trypan blue staining.Results:A case of NRDS with ABCA3 gene complex heterozygous mutation（c.3997₃998del and c.3137C>T）was Confirmed.PBMCs adhered to the wall and growed,then forming clones after infecting with Sendai virus.When the cells growed to 70%,they were continuously passaged.The cell density covered 70-80%of the surface of the culture container,and Y-27632 was added to the culture medium to promote adhesion and growth of iPSCs in order to forma single cell layer covering the surface of the culture container.When the cells growed to 70%,continued to be passaged to P40.Four pluripotency markers including OCT4,Sox2,Nanog and SSEA4 of iwere positive expression in iPSCs.The three markers Expressing pluripotency of iPSCs were tested,including TRA-1-60 positive cells 95.5%,SSEA-4 positive cells 97.88%,and OCT4 positive cells 98.27%,all of which were much higher than 70%,Expression of dry factors including DPPA5,Nanog,OCT3/4,and Sox2 in IPSCs wasmuch higher than that in myocardial cells.It was proved that AFP,Brachyury and PAX-6 were positive expression in endoderm,mesoderm and ectoderm respectively.The analysis of chromosome karyotype,ABCA3 mutation site,and STR site showed that iPSCs were the same as patient with NRDS.There was no Sendai virus residue or pathogenic microbial contamination in iPSCs.IPSCs had a higher production after recovery.Conclusions:This study found complex heterozygous mutations（c.3997-3998del and c.3137C>T）of ABCA3 in the Chinese NRDS.This study successfully constructed a cell line of iPSCsderived from a patient carryingcomplex heterozygous mutations of ABCA3 in NRDS.This study found complex heterozygous mutations（c.3997-3998del and c.3137C>T）of ABCA3 in the Chinese NRDS population.This study may lay the foundation for the further preparation of cells which meet clinical need and are suitable forcell therapy of NRDS carrying complex heterozygous mutationsof ABC A3.