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Glycoproteomic analysis of the changes in protein N-glycosylation during neuronal differentiation in human-induced pluripotent stem cells and derived neuronal cells.

ABSTRACT: N-glycosylation of glycoproteins, a major post-translational modification, plays a crucial role in various biological phenomena. In central nervous systems, N-glycosylation is thought to be associated with differentiation and regeneration; however, the state and role of N-glycosylation in neuronal differentiation remain unclear. Here, we conducted sequential LC/MS/MS analyses of tryptic digest, enriched glycopeptides, and deglycosylated peptides of proteins derived from human-induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells, which were used as a model of neuronal differentiation. We demonstrate that the production profiles of many glycoproteins and their glycoforms were altered during neuronal differentiation. Particularly, the levels of glycoproteins modified with an N-glycan, consisting of five N-acetylhexosamines, three hexoses, and a fucose (HN5H3F), increased in dopaminergic neuron-rich cells (DAs). The N-glycan was deduced to be a fucosylated and bisected biantennary glycan based on product ion spectra. Interestingly, the HN5H3F-modified proteins were predicted to be functionally involved in neural cell adhesion, axon guidance, and the semaphorin-plexin signaling pathway, and protein modifications were site-selective and DA-selective regardless of protein production levels. Our integrated method for glycoproteome analysis and resultant profiles of glycoproteins and their glycoforms provide valuable information for further understanding the role of N-glycosylation in neuronal differentiation and neural regeneration.

SUBMITTER: Kimura K

PROVIDER: S-EPMC8160270 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionHuman induced pluripotent stem cells (hiPSCs) have been derived from various somatic cell types. Granulosa cells, a group of cells which surround oocytes and are obtained from the (normally discarded) retrieved egg follicles of women undergoing infertility treatment, are a possible cell source for induced pluripotent stem cell (iPSC) generation. Here, we explored the possibility of using human granulosa cells as a donor cell type for iPSC reprogramming, and compared granulosa cell-derived iPSCs (iGRAs) with those derived from other cell sources, to determine the potential ability of iGRA differentiation.MethodsGranulosa cells were collected from egg follicles retrieved from women undergoing infertility treatment. After short-term culture, the granulosa cells derived from different patients were mixed in culture, and infected with retroviruses encoding reprogramming factors. The resulting iPSC clones were selected and subjected to microsatellite DNA analysis to determine their parental origin. IGRAs were subjected to RT-PCR, immunofluorescence staining, and in vitro and in vivo differentiation assays to further establish their pluripotent characteristics.ResultsMicrosatellite DNA analysis was used to demonstrate that hiPSCs with different parental origins can be simultaneously reprogrammed by retroviral transfection of a mixed human granulosa cell population obtained from multiple individuals. The iGRAs resemble human embryonic stem cells (hESCs) in many respects, including morphological traits, growth requirements, gene and marker expression profiles, and in vitro and in vivo developmental propensities. We also demonstrate that the iGRAs express low levels of NLRP2, and differentiating iGRAs possess a biased differentiation potential toward the trophoblastic lineage. Although NLRP2 knockdown in hESCs promotes trophoblastic differentiation of differentiating hESCs, it does not result in exit from pluripotency. These results imply that NLRP2 may play a role in regulating the trophoblastic differentiation of human pluripotent stem cells.ConclusionsThese findings provide a means of generating iPSCs from multiple granulosa cell populations with different parental origins. The ability to generate iPSCs from granulosa cells not only enables modeling of infertility-associated disease, but also provides a means of identifying potential clinical interventions through iPSC-based drug screening.

Project description:BACKGROUND:Premature ovarian insufficiency (POI) is a common disease in reproductive women. The pathogenesis of POI is not clear, although it is known that it involves the disorder of oocyte differentiation and development. The introduction of reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) offers a unique opportunity to study many aspects of POI from cell differentiation in vitro that could ultimately lead to novel drug development and testing to help treat the disorder. METHODS:The fibroblasts from POI patients, including fragile X syndrome, abnormal karyotype (45, X; 45, X/46, XX; 45, XO and 47, XXX), and the gene mutation (FIGLA and GDF9) were reprogrammed to pluripotency status by retroviral transduction using defined factors. The morphology, growth characteristics, gene expression profiles, epigenetic status, and in vitro and in vivo differentiation potential of the POI-1-iPSCs (from fragile X syndrome) were analyzed. Then, POI-1-iPSCs were induced to differentiation into primordial germ cells (PGCs) with DNA methyltransferase inhibitors. RESULTS:The iPSCs were successfully generated from POI patients' fibroblasts. The formed iPS clones have the same characteristics of human ESCs. POI-1-iPSCs were successfully generated with germline competence. The POI-1-iPSCs, with genotypes of fragile X syndrome, can be induced to differentiation into PGCs with high efficiency under our culture system by DNA demethylation. This study proved that disease-specific iPSC lines derived from POI patients could be generated and successfully differentiated into PGCs. CONCLUSIONS:We established some novel, systemic cell models for the studying of the pathogenesis of POI patients. Second, DNA demethylation may accelerate the induction of human PGCs from iPSCs in vitro and the conclusion needs further exploration. This represents an important step in the novel approach for the study of the pathophysiology and potential egg resource for POI patients.

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Glycoproteomic analysis of the changes in protein N-glycosylation during neuronal differentiation in human-induced pluripotent stem cells and derived neuronal cells.

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