Biology subjects

Zeevaert, K.

Publications and source records attributed to Zeevaert, K..

7 recordsLinked to original sources

bioRxiv2026

Primed-to-naive conversion of pluripotent stem cells can be tracked by specific DNA methylation changes for optimized culture conditions

During early embryonic development, cells transition from naive to primed pluripotent state. Various culture conditions have been established to revert primed cells back to naive state, to increase differentiation potential and to reset epigenetic abnormalities. In this study, we modified culture conditions to allow primed-to-naive conversion under feeder-independent and normoxic conditions (FINO medium), which exemplified the need for a quantitative measure of pluripotent states. DNA methylation (DNAm) profiling revealed extensive hypomethylation at naive state, but also significant gains of methylation at specific sites in the genome. We demonstrate that DNAm patterns can be used to benchmark culture protocols. Furthermore, we developed a naive-score based on DNAm at two genomic sites, which can be analyzed by digital PCR to monitor transition between pluripotent states. Our study describes a simplified culture protocol for primed-to-naive conversion, offers insights into the specific DNAm changes, and introduces a robust DNAm-based biomarker to track this process effectively.

cell biology↗

bioRxiv2025

Microgels enable iPSCs to assemble, expand, and differentiate into organoids -- from sizable to high throughput

Organoid research holds tremendous potential for personalized medicine and drug development. However, current limitations include reproducibility issues largely due to the use of biologically derived materials, which are prone to batch-to-batch variations. Here, we report a new technology for human induced pluripotent stem cell (iPSC)-based organoid production with iPSC expansion and differentiation in the same construct in a reproducible and scalable manner, compatible with high-throughput automation. Chemically defined poly(ethylene glycol) (PEG)-based microgels are produced via parallelized step-emulsification microfluidics, enabling scalable production. This approach leverages the self-organization of iPSCs with microgels to build three-dimensional constructs, driven by robust cell-material interactions achieved through vitronectin-coated PEG microgels. This technology allows the iPSCs to expand and retain their pluripotency, after which they can be differentiated into the three germ layers, providing a suitable platform for organoid differentiation. This was further extended by differentiation into cardiac organoids and retinal photoreceptors to demonstrate two exemplary tissues.

bioengineering↗

bioRxiv2025

NSD1 governs H3K36me2-mediated DNA methylation and drives differentiation of human iPSCs by regulating HIDEN lncRNA expression

Epigenetic regulatory mechanisms, which include histone modifications and DNA methylation, play a central role in development and aging. Dimethylation of H3K36, deposited mainly by the histone methyltransferase NSD1, occurs predominantly in intergenic regions and recruits the DNA methyltransferase DNMT3A to facilitate DNA methylation. Haploinsufficiency of NSD1 results in the overgrowth disorder Sotos syndrome in vivo, which is associated with aberrant DNA methylation signatures and an enhanced epigenetic age. To understand the mechanisms by which NSD1 may regulate development, differentiation and diseases, we generated human iPSC lines deficient in functional NSD1 (NSD1-KO). NSD1-KO cells exhibit a substrate-specific decrease in proliferation, reduced H3K36me2 levels and extensive DNA hypomethylation. Notably, the loss of functional NSD1 altered the differentiation potential of iPSCs, with aberrant endodermal and mesodermal lineage commitment. Further analysis revealed that NSD1 might drive endodermal differentiation by regulating the expression of an endodermal lncRNA HIDEN by mechanisms independent of the regulation of DNA methylation. Our NSD1-KO iPSC lines partially recapitulate the DNA methylation defects associated with NSD1-related disorders. Additionally, we uncovered a novel mechanism by which NSD1 may regulate endodermal differentiation of human iPSCs.

cell biology↗

bioRxiv2024

YAP1 Contributes to The Development of Contractile Force and Sarcomere Maturation in Human Pluripotent Stem Cell Derived Cardiomyocytes

BackgroundPerspective regenerative therapies for injured myocardium focus on reactivating developmental processes to regenerate damaged contractile tissue. In animal models, the Hippo pathway was shown to improve heart regeneration after myocardial infarction, possibly by expanding the pool of cardiomyocytes. We hypothesized that activating the Hippo pathways downstream effector, Yes Associated Protein (YAP1), may have effects beyond promoting proliferation in human cardiomyocytes. We have therefore investigated how YAP1 influences human cardiomyocyte maturation, sarcomere structure, electrophysiological properties, their response to mechanical stimuli, intracellular Ca2+'' dynamics and force development using models of cardiomyocytes derived from pluripotent stem cells. MethodsWe employed genetic models of YAP1 deficient human embryonic and induced pluripotent stem cells, cardiomyocyte differentiation, compliant cultivation substrates, mechanical actuation, ECM deposition, super resolution microscopy, electrophysiological measurements and engineered heart tissues (EHTs) to characterize the effects of YAP1 deficiency on cardiomyocytes during maturation. We also used full length YAP1 re- expression to rescue the effects of YAP1 deficiency in contracting cardiomyocytes. ResultsYAP1 contributes to cardiomyocyte maturation, participates in the formation and alignment of myofibrils, as well as in the maturation of electrophysiological properties. The net effect of YAP1 deficiency in cardiomyocytes is the inability to respond to physiological stimuli by compensatory growth resulting in reduced force development. Additionally, YAP1 reactivation in contracting cardiomyocytes leads to rescue of myofibril maturation. ConclusionsThis research demonstrates that YAP1 activity is essential to promote cardiomyocyte maturation, contractility, and response to regeneration inducing stimuli.

cell biology↗

bioRxiv2024

Substrate elasticity does not impact on DNA methylation changes during differentiation of pluripotent stem cells

Substrate elasticity may direct cell-fate decisions of stem cells. However, it is largely unclear how matrix stiffness impacts on differentiation of induced pluripotent stem cells (iPSCs) and if this is also reflected by epigenetic modifications. We have therefore cultured iPSCs on tissue culture plastic (TCP) and polydimethylsiloxane (PDMS) with different Youngs modulus (0.2 kPa, 16 kPa, or 64 kPa) to investigate the sequel on growth and differentiation towards endoderm, mesoderm, and ectoderm. Immunofluorescence and gene expression of canonical differentiation markers was hardly affected by the substrates. Notably, when we analyzed DNA methylation profiles of undifferentiated iPSCs or after three-lineage differentiation, we did not see any significant differences on the three different PDMS elasticities. Only when we compared DNA methylation profiles on PDMS-substrates versus TCP, we observed epigenetic differences, particularly upon mesodermal differentiation. Taken together, stiffness of PDMS-substrates did not impact on directed differentiation of iPSCs, whereas the moderate epigenetic differences on TCP might also be attributed to other chemical parameters.

cell biology↗

bioRxiv2022

YAP1 is essential for self-organized differentiation of pluripotent stem cells

The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. It has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human induced pluripotent stem cells (iPSCs) remains largely unclear. To gain better insight into the function of YAP1 in these early cell-fate decisions, we generated iPSC lines with YAP1 knockout (YAP-/-) with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. In YAP-/- iPSCs the expression of several YAP1 targets changed and NODAL, which is an important regulator of cell differentiation, was upregulated. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies toward endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP-/- lines. Notably, in self-organized embryoid bodies (EBs) germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and in tendency also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the non-directed self-organization of EBs, and this is at least partly attributed to activation of the NODAL pathway.

cell biology↗

bioRxiv2020

The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 {micro}m from the outer rim of iPSC colonies. Single- cell RNA-sequencing demonstrated that OCT4high subsets have pronounced up-regulation of the TGF-{beta} pathway, particularly of NODAL and its inhibitor LEFTY, at the rim of the colonies. Furthermore, calcium-dependent cell-cell interactions were found to be relevant for the self-organization. Interestingly, after 5 to 7 days, the iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size, without any enzymatic or mechanical treatment. Within the early 3D aggregates, the radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

cell biology↗