Abstract
BackgroundAutosomal dominant osteopetrosis type II (ADO2) is a rare human genetic disease that has been broadly studied as an important osteopetrosis model; however, there are no disease-specific induced pluripotent stem cells (ADO2-iPSCs) that may be valuable for understanding the pathogenesis and may be a potential source of cells for autologous cell-based therapies.MethodsTo generate the first human ADO2-iPSCs from a Chinese family with ADO2 and to identify their characteristics, blood samples were collected from the proband and his parents and were used for genotyping by whole-exome sequencing (WES); the urine-derived cells of the proband were reprogrammed with episomal plasmids that contained transcription factors, such as KLF4, OCT4, c-MYC, and SOX2. The proteome-wide protein quantification and lysine 2-hydroxyisobutyrylation detection of the ADO2-iPSCs and normal control iPSCs (NC-iPSCs) were performed by high-resolution LC-MS/MS and bioinformatics analysis.ResultsWES with filtering strategies identified a mutation in CLCN7 (R286W) in the proband and his father, which was absent in the proband’s mother and the healthy controls; this was confirmed by Sanger sequencing. The ADO2-iPSCs were successfully generated, which carried a normal male karyotype (46, XY) and the mutation of CLCN7 (R286W); the ADO2-iPSCs positively expressed alkaline phosphatase and other surface markers; and no vector and transgene were detected. The ADO2-iPSCs could differentiate into all three germ cell layers, both in vitro and in vivo. The proteomic profiling revealed similar expression of pluripotency markers in the two cell lines and identified 7405 proteins and 3664 2-hydroxyisobutyrylated peptides in 1036 proteins in the ADO2-iPSCs.ConclusionsOur data indicated that the mutation CLCN7 (R286W) may be a cause of the osteopetrosis family. The generated vector-free and transgene-free ADO2-iPSCs with known proteomic characteristics may be valuable for personalized and cell-based regenerative medicine in the future.
Highlights
Autosomal dominant osteopetrosis type II (ADO2) is a rare human genetic disease that has been broadly studied as an important osteopetrosis model; there are no disease-specific induced pluripotent stem cells (ADO2-iPSCs) that may be valuable for understanding the pathogenesis and may be a potential source of cells for autologous cell-based therapies
The patients with more severe conditions were commonly observed as autosomal recessive osteopetrosis (ARO), and those with mild conditions were more commonly found in adults with autosomal dominant osteopetrosis type II (ADO2) [3]
Genotyping and the generation of ADO2-iPSCs Genotyping of the osteopetrotic family The exomes of the proband and his parents from the ADO2 family were captured and sequenced
Summary
Autosomal dominant osteopetrosis type II (ADO2) is a rare human genetic disease that has been broadly studied as an important osteopetrosis model; there are no disease-specific induced pluripotent stem cells (ADO2-iPSCs) that may be valuable for understanding the pathogenesis and may be a potential source of cells for autologous cell-based therapies. Allogeneic hematopoietic stem cell transplantation (HSCT) treatments have been chosen for the treatment of severe osteopetrosis, which results in 73% of patients achieving 5 years of disease-free survival [4] This kind of treatment has been greatly improved over the past few years, but the engraftment of mesenchymal stem cells from donors may have unexpected difficulties, allogeneic HSCT is still a dangerous procedure with other kinds of toxicities and is limited by the requirement of a matched donor [5, 6]. It is important that the urine can be obtained by a noninvasive procedure, and patient urinary iPSCs have been found valuably in disease modeling and regenerative medicine [9]
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