Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells

Aalders, Jan G.; Vreken, Natasja Van den; Popovic, Mina; Mishra, Swati; Heindryckx, Björn; Hengel, Jolanda van

doi:10.1007/s11626-019-00413-9

Cited by 4 publications

(3 citation statements)

References 23 publications

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“…The petri dishes were pre-treated with Matrigel, and the medium was changed daily. After culturing for 4–5 days, the iPSCs were digested, and the suspended cell clusters were collected and subcultured at a ratio of 1:6 at 37°C, 5% CO 2 (Aalders et al, 2019 ).…”

Section: Methodsmentioning

confidence: 99%

The Study of Alternative Splicing Events in Human Induced Pluripotent Stem Cells From a Down's Syndrome Patient

Yang

Cai

et al. 2021

Front. Cell Dev. Biol.

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Down's syndrome (DS) is one of the most commonly known disorders with multiple congenital disabilities. Besides severe cognitive impairment and intellectual disability, individuals with DS also exhibit additional phenotypes of variable penetrance and severity, with one or more comorbid conditions, including Alzheimer's disease, congenital heart disease, or leukemia. Various vital genes and regulatory networks had been studied to reveal the pathogenesis of the disease. Nevertheless, very few studies have examined alternative splicing. Alternative splicing (AS) is a regulatory mechanism of gene expression when making one multi-exon protein-coding gene produce more than one unique mature mRNA. We employed the GeneChip Human Transcriptome Array 2.0 (HTA 2.0) for the global gene analysis with hiPSCs from DS and healthy individuals. Examining differentially expressed genes (DEGs) in these groups and focusing on specific transcripts with AS, 466 up-regulated and 722 down-regulated genes with AS events were identified. These genes were significantly enriched in biological processes, such as cell adhesion, cardiac muscle contraction, and immune response, through gene ontology (GO) analysis of DEGs. Candidate genes, such as FN1 were further explored for potentially playing a key role in DS. This study provides important insights into the potential role that AS plays in DS.

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Section: Methodsmentioning

confidence: 99%

The Study of Alternative Splicing Events in Human Induced Pluripotent Stem Cells From a Down's Syndrome Patient

Yang

Cai

et al. 2021

Front. Cell Dev. Biol.

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“…A1413302) and cultured in Essential 8 medium (Cat No. A1517001) supplemented with P/S with a seeding density of 2.1 × 10 4 cells/cm 2 26 . The H9 human embryonic stem cell (hESC) line (WA09, WiCell, feeder free cultures were obtained via prof. C. Verfaillie, KULeuven, Belgium) was cultured feeder-free in Essential 8 medium under the same conditions as the MFS and corrected lines and was used as control.…”

Section: Methodsmentioning

confidence: 99%

Effects of fibrillin mutations on the behavior of heart muscle cells in Marfan syndrome

Aalders

Léger

Meeren

et al. 2020

Sci Rep

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Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by pathogenic variants in the fibrillin-1 (FBN1) gene. Myocardial dysfunction has been demonstrated in MFS patients and mouse models, but little is known about the intrinsic effect on the cardiomyocytes (CMs). In this study, both induced pluripotent stem cells derived from a MFS-patient and the line with the corrected FBN1 mutation were differentiated to CMs. Several functional analyses are performed on this model to study MFS related cardiomyopathy. Atomic force microscopy revealed that MFS CMs are stiffer compared to corrected CMs. The contraction amplitude of MFS CMs is decreased compared to corrected CMs. Under normal culture conditions, MFS CMs show a lower beat-to-beat variability compared to corrected CMs using multi electrode array. Isoproterenol-induced stress or cyclic strain demonstrates lack of support from the matrix in MFS CMs. This study reports the first cardiac cell culture model for MFS, revealing abnormalities in the behavior of MFS CMs that are related to matrix defects. Based on these results, we postulate that impaired support from the extracellular environment plays a key role in the improper functioning of CMs in MFS.

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“…Naïve human embryonic stem cells (hESCs) cultured on CF-1 irradiated Mouse Embryonic Fibroblasts (MEFs) (A34180, Gibco TM ) were kindly provided by the group of Björn Heindryckx, Ghent University (Duggal et al, 2015) 28 and converted to feeder-free naïve-like human embryonic stem cells with RSeT Feeder-Free medium (STEMCELL TM technologies) according to manufacturer's instructions with an adapted in-house protocol (derived from Aalders, et al 2019) 29 described in the Supplementary Material.…”

Section: Human Embryonic Stem Cells (Hescs)mentioning

confidence: 99%

A novel neurodevelopmental syndrome caused by loss-of-function of the Zinc Finger Homeobox 3 (ZFHX3) gene

Baca

Jacobs

Vantomme

et al. 2023

Preprint

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Neurodevelopmental disorders (NDDs) result from impaired development and functioning of the brain. Here, we identify loss-of-function variation in ZFHX3 as a novel cause for syndromic intellectual disability (ID). ZFHX3, previously known as ATBF1, is a zinc-finger homeodomain transcription factor involved in multiple biological processes including cell differentiation and tumorigenesis. Through international collaboration, we collected clinical and morphometric data (Face2Gene) of 41 individuals with protein truncating variants (PTVs) or (partial) deletions of ZFHX3. We used data mining, RNA and protein analysis to identify the subcellular localization and spatiotemporal expression of ZFHX3 in multiple in vitro models. We identified the DNA targets of ZFHX3 using ChIP seq. Immunoprecipitation followed by mass spectrometry indicated potential binding partners of endogenous ZFHX3 in neural stem cells that were subsequently confirmed by reversed co-immunoprecipitation and western blot. We evaluated a DNA methylation profile associated with ZFHX3 haploinsufficiency using DNA methylation analysis on whole blood extracted DNA of six individuals with ZFHX3 PTVs and four with a (partial) deletion of ZFHX3. A reversed genetic approach characterized the ZFHX3 orthologue in Drosophila melanogaster. Loss-of-function variation of ZFHX3 consistently associates with (mild) ID and/or behavioural problems, postnatal growth retardation, feeding difficulties, and recognizable facial characteristics, including the rare occurrence of cleft palate. Nuclear abundance of ZFHX3 increases during human brain development and neuronal differentiation In neural stem cells and SH-SY5Y cells, ZFHX3 interacts with the chromatin remodelling BRG1/Brm-associated factor complex and the cleavage and polyadenylation complex. In line with a role for chromatin remodelling, ZFHX3 haploinsufficiency associates with a specific DNA methylation profile in leukocyte-derived DNA. The target genes of ZFHX3 are implicated in neuron and axon development. In Drosophila melanogaster, zfh2, considered to be the ZFHX3 orthologue, is expressed in the third instar larval brain. Ubiquitous and neuron-specific knockdown of zfh2 results in adult lethality underscoring a key role for zfh2 in development and neurodevelopment. Interestingly, ectopic expression of zfh2 as well as ZFHX3 in the developing wing disc results in a thoracic cleft phenotype. Collectively, our data shows that loss-of-function variants in ZFHX3 are a cause of syndromic ID, that associates with a specific DNA methylation profile. Furthermore, we show that ZFHX3 participates in chromatin remodelling and mRNA processing.

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Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells

Cited by 4 publications

References 23 publications

The Study of Alternative Splicing Events in Human Induced Pluripotent Stem Cells From a Down's Syndrome Patient

The Study of Alternative Splicing Events in Human Induced Pluripotent Stem Cells From a Down's Syndrome Patient

Effects of fibrillin mutations on the behavior of heart muscle cells in Marfan syndrome

A novel neurodevelopmental syndrome caused by loss-of-function of the Zinc Finger Homeobox 3 (ZFHX3) gene

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