Cargando…

Genotyping, generation and proteomic profiling of the first human autosomal dominant osteopetrosis type II-specific induced pluripotent stem cells

BACKGROUND: Autosomal 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 an...

Descripción completa

Detalles Bibliográficos
Autores principales: Ou, Minglin, Li, Chunhong, Tang, Donge, Xue, Wen, Xu, Yong, Zhu, Peng, Li, Bo, Xie, Jiansheng, Chen, Jiejing, Sui, Weiguo, Yin, Lianghong, Dai, Yong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693165/
https://www.ncbi.nlm.nih.gov/pubmed/31412925
http://dx.doi.org/10.1186/s13287-019-1369-8
_version_ 1783443657120022528
author Ou, Minglin
Li, Chunhong
Tang, Donge
Xue, Wen
Xu, Yong
Zhu, Peng
Li, Bo
Xie, Jiansheng
Chen, Jiejing
Sui, Weiguo
Yin, Lianghong
Dai, Yong
author_facet Ou, Minglin
Li, Chunhong
Tang, Donge
Xue, Wen
Xu, Yong
Zhu, Peng
Li, Bo
Xie, Jiansheng
Chen, Jiejing
Sui, Weiguo
Yin, Lianghong
Dai, Yong
author_sort Ou, Minglin
collection PubMed
description BACKGROUND: Autosomal 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. METHODS: To 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. RESULTS: WES 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. CONCLUSIONS: Our 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13287-019-1369-8) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-6693165
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-66931652019-08-16 Genotyping, generation and proteomic profiling of the first human autosomal dominant osteopetrosis type II-specific induced pluripotent stem cells Ou, Minglin Li, Chunhong Tang, Donge Xue, Wen Xu, Yong Zhu, Peng Li, Bo Xie, Jiansheng Chen, Jiejing Sui, Weiguo Yin, Lianghong Dai, Yong Stem Cell Res Ther Research BACKGROUND: Autosomal 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. METHODS: To 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. RESULTS: WES 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. CONCLUSIONS: Our 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13287-019-1369-8) contains supplementary material, which is available to authorized users. BioMed Central 2019-08-14 /pmc/articles/PMC6693165/ /pubmed/31412925 http://dx.doi.org/10.1186/s13287-019-1369-8 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Ou, Minglin
Li, Chunhong
Tang, Donge
Xue, Wen
Xu, Yong
Zhu, Peng
Li, Bo
Xie, Jiansheng
Chen, Jiejing
Sui, Weiguo
Yin, Lianghong
Dai, Yong
Genotyping, generation and proteomic profiling of the first human autosomal dominant osteopetrosis type II-specific induced pluripotent stem cells
title Genotyping, generation and proteomic profiling of the first human autosomal dominant osteopetrosis type II-specific induced pluripotent stem cells
title_full Genotyping, generation and proteomic profiling of the first human autosomal dominant osteopetrosis type II-specific induced pluripotent stem cells
title_fullStr Genotyping, generation and proteomic profiling of the first human autosomal dominant osteopetrosis type II-specific induced pluripotent stem cells
title_full_unstemmed Genotyping, generation and proteomic profiling of the first human autosomal dominant osteopetrosis type II-specific induced pluripotent stem cells
title_short Genotyping, generation and proteomic profiling of the first human autosomal dominant osteopetrosis type II-specific induced pluripotent stem cells
title_sort genotyping, generation and proteomic profiling of the first human autosomal dominant osteopetrosis type ii-specific induced pluripotent stem cells
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693165/
https://www.ncbi.nlm.nih.gov/pubmed/31412925
http://dx.doi.org/10.1186/s13287-019-1369-8
work_keys_str_mv AT ouminglin genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT lichunhong genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT tangdonge genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT xuewen genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT xuyong genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT zhupeng genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT libo genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT xiejiansheng genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT chenjiejing genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT suiweiguo genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT yinlianghong genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells
AT daiyong genotypinggenerationandproteomicprofilingofthefirsthumanautosomaldominantosteopetrosistypeiispecificinducedpluripotentstemcells