Epigenetic Biomarker to Support Classification into Pluripotent and Non-Pluripotent Cells

Lenz, Michael; Goetzke, Roman; Schenk, Arne; Schubert, Claudia; Veeck, Jürgen; Hemeda, Hatim; Koschmieder, Steffen; Zenke, Martin; Schuppert, Andreas; Wagner, Wolfgang

doi:10.1038/srep08973

Cited by 46 publications

(57 citation statements)

References 41 publications

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“…iPS cells were generated from BM hematopoietic progenitors with OCT4, SOX2, c‐MYC, and KLF4 in Sendai virus vectors. iPS cells were pluripotent by Epi‐Pluri‐Score test , expression of pluripotency markers and three germ layer differentiation potential (Supporting Information Fig. 1A‐1C).…”

Section: Resultsmentioning

confidence: 99%

“…Data are presented as mean or median 6 standard deviation (SD). Statistical significance was analyzed using two-tailed, unpaired Student t test (GraphPad Prism version 6) and differences were considered significant (*) when p < .05, very significant (**) when p < .005, and extremely significant (***) when p < .001. cells were pluripotent by Epi-Pluri-Score test [34], expression of pluripotency markers and three germ layer differentiation potential (Supporting Information Fig. 1A-1C).…”

Section: Statistical Analysesmentioning

confidence: 99%

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Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells

et al. 2017

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Human induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers, including hematopoietic stem cells and their progeny. Interferon regulatory factor 8 (IRF8) is a transcription factor, which acts in hematopoiesis as lineage determining factor for myeloid cells, including dendritic cells (DC). Autosomal recessive or dominant IRF8 mutations occurring in patients cause severe monocytic and DC immunodeficiency. To study IRF8 in human hematopoiesis we generated human IRF82/2 iPS cells and IRF82/2 embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing. Upon induction of hematopoietic differentiation, we demonstrate that IRF8 is dispensable for iPS cell and ES cell differentiation into hemogenic endothelium and for endothelial-to-hematopoietic transition, and thus development of hematopoietic progenitors. We differentiated iPS cell and ES cell derived progenitors into CD1411 cross-presenting cDC1 and CD1c1 classical cDC2 and CD3031 plasmacytoid DC (pDC). We found that IRF8 deficiency compromised cDC1 and pDC development, while cDC2 development was largely unaffected. Additionally, in an unrestricted differentiation regimen, IRF82/2 iPS cells and ES cells exhibited a clear bias toward granulocytes at the expense of monocytes. IRF82/2 DC showed reduced MHC class II expression and were impaired in cytokine responses, migration, and antigen presentation. Taken together, we engineered a human IRF8 knockout model that allows studying molecular mechanisms of human immunodeficiencies in vitro, including the pathophysiology of IRF8 deficient DC. STEM CELLS 2017;35:898-908 SIGNIFICANCE STATEMENTPluripotent stem cells and CRISPR/Cas9n technology are particularly well suited for engineering cells to study the impact of specific factors on cell development, including antigen presenting dendritic cells (DC). So far, DC research was limited to primary cell samples obtained for example, from mice or men. In the mouse system, genetically modified DC are readily obtained by using transgenic, knockout, and knockin mice. In the human system studies with mutated DC relied on patients harboring specific mutations and there was a paucity of techniques for genetic engineering directly in human cells. Induced pluripotent stem (iPS) cell and CRISPR/ Cas9n technology now allow to overcome these limitations. Here, we generated interferon regulatory factor 8 (IRF8) knockout human iPS cells and ES cells, and IRF82/2 DC derived thereof. We show that IRF82/2 cells recapitulate the phenotype of individuals with an IRF8 loss of function mutation. Our IRF82/2 iPS cells and ES cells provide a platform to study IRF8 deficient DC subset specification and DC function independent of donor variation or availability. In summary, our IRF82/2 iPS cells and ES cells represent a valid and powerful model to elucidate mechanisms of human DC development and functional diversity.

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Section: Resultsmentioning

confidence: 99%

Section: Statistical Analysesmentioning

confidence: 99%

Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells

et al. 2017

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“…Pluripotency was assessed by immunophenotypic analysis, multilineage differentiation, and Epi-Pluri-Score (Lenz et al., 2015). iPSCs were cultured in 6-well plates coated with 0.5 μg/cm 2 of vitronectin (STEMCELL Technologies) and maintained in iPS-Brew medium (Miltenyi Biotec) supplemented with 1% penicillin/streptomycin (Life Technologies).…”

Section: Methodsmentioning

confidence: 99%

Surface Topography Guides Morphology and Spatial Patterning of Induced Pluripotent Stem Cell Colonies

et al. 2017

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SummaryThe relevance of topographic cues for commitment of induced pluripotent stem cells (iPSCs) is largely unknown. In this study, we demonstrate that groove-ridge structures with a periodicity in the submicrometer range induce elongation of iPSC colonies, guide the orientation of apical actin fibers, and direct the polarity of cell division. Elongation of iPSC colonies impacts also on their intrinsic molecular patterning, which seems to be orchestrated from the rim of the colonies. BMP4-induced differentiation is enhanced in elongated colonies, and the submicron grooves impact on the spatial modulation of YAP activity upon induction with this morphogen. Interestingly, TAZ, a YAP paralog, shows distinct cytoskeletal localization in iPSCs. These findings demonstrate that topography can guide orientation and organization of iPSC colonies, which may affect the interaction between mechanosensors and mechanotransducers in iPSCs.

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“…This work has received much attention and has moved towards commercial application (TaqMan® hPSC Scorecard™ Assay provided by ThermoFisher Scientific in a revised version [9]). Furthermore, pluripotency assessment using computational models built on DNA methylation states of cells have already been implemented and could potentially be combined with PluriTest technologies [11]. The complexity of histone tail modifications and the Bhistone code^ [44] of pluripotent cells has so far not been utilized for hPSC pluripotency assessment and quality control.…”

Section: Epigenetic Marks For Pluripotency Assessmentmentioning

confidence: 99%

Utilizing Regulatory Networks for Pluripotency Assessment in Stem Cells

et al. 2016

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Pluripotency is a term in cell biology describing a unique state present in distinct stem cell lines, which were either established from the inner cell mass of the mammalian embryo or derived from somatic cells that have been reprogrammed to induced pluripotent stem cells. Pluripotent stem cells are continuously self-renewing, and their differentiation capacity enables them to develop into all derivatives of the three germ layers of a gastrulating embryo (endoderm, ectoderm, mesoderm). Both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) are virtually indistinguishable, at least based on their global RNA expression patterns. Yet, after these in vitro cell cultures have been generated, the cell lines' pluripotent properties may change considerably on the genetic and/or epigenetic level as a consequence of long-term propagation. Among other unphysiological changes, cell lines might acquire aneuploidies, loose physiological imprinting marks, or develop differentiation biases favoring one cell lineage over the other. As a result, stem cell researchers have to continuously monitor each stem cell line's integrity, transcriptional profile, and functional properties. Regulatory transcription factors, proteinprotein interactions, and signaling networks govern the pluripotent state. As a consequence, emerging small-and largescale perturbations to these gene regulatory networks mediate the outlined unfavorable changes to the pluripotent phenotype. Here, we describe a reliable bioinformatic framework called PluriTest for confirmation and assessment of pluripotency as an animal-free, fast, and inexpensive way based on genome-wide transcriptional RNA profiles from microarrays. Additionally, we discuss future developments using RNA expression profiling for pluripotency assessment.

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Epigenetic Biomarker to Support Classification into Pluripotent and Non-Pluripotent Cells

Cited by 46 publications

References 41 publications

Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells

Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells

Surface Topography Guides Morphology and Spatial Patterning of Induced Pluripotent Stem Cell Colonies

Utilizing Regulatory Networks for Pluripotency Assessment in Stem Cells

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