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

Mabrouk, Mohamed H Elsafi; Goetzke, Roman; Abagnale, Giulio; Yesilyurt, Burcu; Salz, Lucia; Cypris, Olivia; Glück, Philipp; Liesenfelder, Sven; Zeevaert, Kira; Ma, Zhiyao; Toledo, Marcelo Augusto Szymanski de; Li, Ronghui; Costa, Ivan G.; Lampert, Angelika; Vivek, P.; Schnakenberg, Uwe; Zenke, Martin; Wagner, Wolfgang

doi:10.1016/j.biomaterials.2022.121389

Cited by 18 publications

(18 citation statements)

References 36 publications

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“…Additionally, a novel and innovative approach to control EB size is to use micro-contact printing (µCP) ( Elsafi Mabrouk et al, 2022 ). iPS cell colony size and hence EB size are controlled by the size of the vitronectin area printed on the tissue culture plate.…”

Section: Resultsmentioning

confidence: 99%

“…Briefly, polydimethylsiloxane stamps with circular features of 600 µm in diameter were used to pattern the surface of 6-well tissue culture plates with vitronectin arrays. iPS cells were seeded on patterned plates by LHU and cultured in StemMACS iPS-Brew XF (Miltenyi Biotech) ( Elsafi Mabrouk et al, 2022 ). iPS cell colonies were cultured for up to 11 days and the rate of colony detachment from µprinted arrays was quantified daily.…”

Section: Methodsmentioning

confidence: 99%

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Cell Cluster Sorting in Automated Differentiation of Patient-specific Induced Pluripotent Stem Cells Towards Blood Cells

Toledo

Wanek

et al. 2022

Front. Bioeng. Biotechnol.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Cell Cluster Sorting in Automated Differentiation of Patient-specific Induced Pluripotent Stem Cells Towards Blood Cells

Toledo

Wanek

et al. 2022

Front. Bioeng. Biotechnol.

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“…To differentiate iPSCs into hematopoietic progenitors, we adjusted the protocol from Liu et al [ 44 ]: EBs were generated from iPSCs by self-detachment from microcontact-printed vitronectin spots generated with PDMS stamps [ 45 ]. EBs were carefully resuspended in serum free medium containing 50% IMDM, 50% Ham’s F12, 0.5% BSA, 1% chemically defined lipid concentrate, 2 mM GlutaMAX (all Thermo Fisher Scientific), 400 μM 1-thioglycerol, 50 μg/mL L-ascorbic acid, and 6 μg/mL holo transferrin (all Sigma Aldrich, St. Louis, MO, USA) supplemented with 10 ng/mL FGF-2 (Peprotech, Hamburg, Germany), 10 ng/mL BMP-4 (Miltenyi Biotec), and 10 μM Y-27632 (Abcam, Cambridge, Great Britain).…”

Section: Methodsmentioning

confidence: 99%

Hematopoietic differentiation persists in human iPSCs defective in de novo DNA methylation

et al. 2022

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Background DNA methylation is involved in the epigenetic regulation of gene expression during developmental processes and is primarily established by the DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B). DNMT3A is one of the most frequently mutated genes in clonal hematopoiesis and leukemia, indicating that it plays a crucial role for hematopoietic differentiation. However, the functional relevance of Dnmt3a for hematopoietic differentiation and hematological malignancies has mostly been analyzed in mice, with the specific role for human hematopoiesis remaining elusive. In this study, we therefore investigated if DNMT3A is essential for hematopoietic differentiation of human induced pluripotent stem cells (iPSCs). Results We generated iPSC lines with knockout of either exon 2, 19, or 23 and analyzed the impact of different DNMT3A exon knockouts on directed differentiation toward mesenchymal and hematopoietic lineages. Exon 19−/− and 23−/− lines displayed an almost entire absence of de novo DNA methylation during mesenchymal and hematopoietic differentiation. Yet, differentiation efficiency was only slightly reduced in exon 19−/− and rather increased in exon 23−/− lines, while there was no significant impact on gene expression in hematopoietic progenitors (iHPCs). Notably, DNMT3A−/− iHPCs recapitulate some DNA methylation patterns of acute myeloid leukemia (AML) with DNMT3A mutations. Furthermore, multicolor genetic barcoding revealed growth advantage of exon 23−/− iHPCs in a syngeneic competitive differentiation assay. Conclusions Our results demonstrate that iPSCs with homozygous knockout of different exons of DNMT3A remain capable of mesenchymal and hematopoietic differentiation—and exon 23−/− iHPCs even gained growth advantage—despite loss of almost the entire de novo DNA methylation. Partial recapitulation of DNA methylation patterns of AML with DNMT3A mutations by our DNMT3A knockout iHPCs indicates that our model system can help to elucidate mechanisms of clonal hematopoiesis.

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“…The iPSC lines were seeded with 10,000 cells/cm 2 in StemMACS iPS-Brew XF with ROCK inhibitor Y-27632 (10 µM) added for the first day. The iPSC growth was restricted to the µCP area with upregulation of pluripotency associated markers at the rim (Elsafi Mabrouk et al, 2022). After six to eight days, the iPSC colonies self-detached spontaneously from the vitronectin-µCP substrates (Elsafi Mabrouk et al, 2022).…”

Section: Spatially Confined Ipsc Colonies and Embryoid Body Formationmentioning

confidence: 99%

“…Furthermore, NODAL seems to play a central role for the cellular organization within iPSC colonies (Tewary et al, 2017; Warmflash et al, 2014). Cells at the outer rim of colonies upregulate pluripotency-associated genes as well as the TGF-β pathway, particularly NODAL and its inhibitor left-right determination factor 1 (LEFTY1) (Elsafi Mabrouk et al, 2022). Another important signaling pathway of early embryo development is the Hippo pathway (Wu and Guan, 2021).…”

Section: Introductionmentioning

confidence: 99%

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

Zeevaert

Goetzke

Mabrouk

et al. 2022

Preprint

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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.

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

Cited by 18 publications

References 36 publications

Cell Cluster Sorting in Automated Differentiation of Patient-specific Induced Pluripotent Stem Cells Towards Blood Cells

Cell Cluster Sorting in Automated Differentiation of Patient-specific Induced Pluripotent Stem Cells Towards Blood Cells

Hematopoietic differentiation persists in human iPSCs defective in de novo DNA methylation

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

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