Generation of an induced pluripotent stem cell line from a patient with surfactant metabolism dysfunction carrying ABCA3 mutations
10.3760/cma.j.cn101070-20230516-00384
- VernacularTitle:携带 ABCA3突变的肺表面活性物质代谢障碍患儿的诱导多能干细胞系构建
- Author:
Zhichen TIAN
1
;
Xin XIE
;
Jinghan CHI
;
Jia CHEN
;
Danhua ZHAO
;
Yanmei HE
;
Xiaojuan YIN
Author Information
1. 中国人民解放军总医院海南医院儿科，三亚 572000
- Keywords:
Infant, newborn;
Respiratory distress syndrome;
Adenosine triphosphate-binding cassette transporter A3;
Induced pluripotent stem cells;
Peripheral blood m
- From:
Chinese Journal of Applied Clinical Pediatrics
2024;39(2):98-103
- CountryChina
- Language:Chinese
-
Abstract:
Objective:Induced pluripotent stem cells (iPSCs) cell lines were established using peripheral blood mononuclear cells (PBMCs) from a patient suffering from neonatal respiratory distress syndrome (NRDS) who carried Adenosine triphosphate-binding cassette transporter A3 ( ABCA3) compound heterozygous mutations. Methods:Cell experimental research.Peripheral venous blood was collected and PBMCs were isolated and cultured in vitro. PBMCs were transfected with non-integrated Sendai vector carrying reprogramming factors.The chromosome karyotypes of the established iPSCs were analyzed.Immunofluorescence and flow cytometry were used to detect pluripotency markers of stem cells and verify their differentiation potential.Sanger sequencing was performed to analyze gene mutations.In addition, short tandem repeat (STR) analysis was performed, polymerase chain reaction(PCR) and agarose gel electrophoresis were used to detect virus residual. Results:Karyotype analysis of established iPSCs cell lines showed normal diploid 46, XY karyotype.Immunofluorescence showed positive staining of stem cell pluripotency markers OCT4, SSEA4, Nanog and Sox2.Flow cytometry was used to detected stem cell pluripotency markers and showed expression of TRA-1-60, SSEA-4 and OCT4.After differentiation into all three germ layers, immunofluorescence was performed to detect ectoderm (Pax-6), mesoderm (Brachyury) and endoderm alpha-fetoprotein markers, and the results showed positive staining, which confirmed that the iPSCs had the potential to differentiate.Sanger sequencing showed c. 3997_3998del and c. 3137C>T compound heterozygous mutations.STR analysis showed they originate from PBMCs, and no Sendai virus residual was detected by PCR and agarose gel electrophoresis.Conclusions:In this study, PBMCs from patient carrying ABCA3 compound heterozygous mutations was used to establish iPSCs cell lines.The research lays a foundation for the study of pathogenesis, therapeutic drug screening and cell therapy of NRDS caused by ABCA3 gene mutations.
