Unknown,Transcriptomics,Genomics,Proteomics

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Cell Fusion Enhances Mesendodermal Differentiation of Human Induced Pluripotent Stem Cells.

ABSTRACT: Human induced pluripotent stem cells (iPS cells) resemble embryonic stem cells and can differentiate into cell derivatives of all three germ layers. However, frequently the differentiation efficiency of iPS cells into some lineages is rather poor. Here, we found that fusion of iPS cells with human hematopoietic stem cells (HSC) enhances iPS cell differentiation. Such iPS hybrids showed a prominent differentiation bias towards hematopoietic lineages but also towards other mesendodermal lineages. Additionally, during differentiation of iPS hybrids expression of early mesendodermal markers - Brachyury (T), MIX1 Homeobox-Like Protein 1 (MIXL1) and Goosecoid (GSC) - appeared with faster kinetics than in parental iPS cells. Following iPS hybrid differentiation there was a prominent induction of NODAL and inhibition of NODAL signaling blunted mesendodermal differentiation. This indicates that NODAL signaling is critically involved in mesendodermal bias of iPS hybrid differentiation. In summary, we demonstrate that iPS cell fusion with HSC prominently enhances iPS differentiation. 11 samples were hybridized GeneChip Human Gene 1.0 ST Arrays (Affymetrix)

ORGANISM(S): Homo sapiens

SUBMITTER: Martin Zenke

PROVIDER: E-GEOD-58457 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Cell fusion enhances mesendodermal differentiation of human induced pluripotent stem cells.

Qin Jie J Sontag Stephanie S Lin Qiong Q Mitzka Saskia S Leisten Isabelle I Schneider Rebekka K RK Wang Xiaoying X Jauch Anna A Peitz Michael M Brüstle Oliver O Wagner Wolfgang W Zhao Robert Chunhua RC Zenke Martin M

Stem cells and development 20140811 23

Human induced pluripotent stem cells (iPS cells) resemble embryonic stem cells and can differentiate into cell derivatives of all three germ layers. However, frequently the differentiation efficiency of iPS cells into some lineages is rather poor. Here, we found that fusion of iPS cells with human hematopoietic stem cells (HSCs) enhances iPS cell differentiation. Such iPS hybrids showed a prominent differentiation bias toward hematopoietic lineages but also toward other mesendodermal lineages. A ...[more]

PMID: 25004077

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Project description:Nodal and Activin are morphogens of the TGFbeta superfamily of signaling molecules that direct differential cell fate decisions in a dose- and distance-dependent manner. During early embryonic development the Nodal/Activin pathway is responsible for the specification of mesoderm, endoderm, node and mesendoderm. In contradiction to this drive towards cellular differentiation, the pathway also plays important roles in the maintenance of self-renewal and pluripotency in embryonic and epiblast stem cells. The molecular basis behind stem cell interpretation of Nodal/Activin signaling gradients and the undertaking of disparate cell fate decisions remains poorly understood. Here, we show that any perturbation of endogenous signaling levels in mouse ES cells leads to their exit from self renewal towards divergent differentiation programs. Increasing Nodal signals above basal levels by direct stimulation with Activin promotes differentiation towards the mesendodermal lineages while repression of signaling with the specific Nodal/Activin receptor inhibitor SB431542 induces trophectodermal differentiation. To address how quantitative Nodal/Activin signals are translated qualitatively into distinct cell fates decisions, we performed chromatin immunoprecipitation of phospho-Smad2 the primary downstream transcriptional factor of the Nodal/Activin pathway followed by massively parallel sequencing and show that phospho-Smad2 binds to and regulates distinct subsets of target genes in a dose-dependent manner. Crucially, Nodal/Activin signaling directly controls the Oct4 master regulator of pluripotency by graded phospho-Smad2 binding in the promoter region. Hence stem cells interpret and carry out differential Nodal/Activin signaling instructions via a corresponding gradient of Smad2 phosphorylation that selectively titrates self-renewal against alternative differentiation programs by direct regulation of distinct target gene subsets and Oct4 expression. Four biological replicates consisting of 4 different passages of E14TG2a ES cells at P20, P21, P23 and P24

Project description:Recent establishment of induced pluripotent stem (iPS) cells opened new avenues for generating human patient-specific stem cell derivatives that can be used for in vitro modeling of human disease, drug development and cell replacement therapy. In this study we analyzed the molecular and functional properties of cardiomyocytes (CM) differentiated from human iPS cells. Clusters of synchronously beating cells were first observed at day 11 of iPS cell differentiation. Beating clusters that were microdissected at day 18 of differentiation expressed high levels of cardiospecific transcripts NKx2.5, alpha-MHC, MLC2v, alpha-actinin and troponin T. Immunocytochemical stainings for alpha-actinin and troponin T revealed cross-striations typical of CM. Functional assessment of iPS cell-derived CM showed that these cells possess intact calcium transients and respond to beta-adrenergic and muscarinic stimulation. Molecular, structural and electrophysiological properties of iPS cell-derived CMs were highly comparable to their human ES cell-derived counterparts at the same differentiation stage. Comparison of global gene expression profiles of human ES and iPS cells and the corresponding microdissected beating areas further confirmed their similarity. We conclude that human iPS cells can differentiate into functional CM, which are indistinguishable from human ES cell-derived CMs and may fulfill the basic requirement for their use in disease modeling, drug screening and future therapeutic applications. Six different experimental groups were included into analysis: undifferentiated human ES cells (1) and undifferentiated human iPS cells (2), human ES cell-derived cardiomyocytes (3) and human iPS cell-derived cardiomyocytes (4) enriched by microdissection of spontaneously contracting embryoid body outgrowths, and human fetal (5) and adult (6) heart tissue. Total RNA samples were prepared from three independent biological replicates in groups 1-4. In groups 5 and 6, single RNA probes were analyzed as three technical replicates.

Project description:Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen, a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified key pathways and regulators of prostatic differentiation including Wnt5a, BPM7, PTEN, and NKx3.1 using E-PZ-iPS-lke cells. Our results suggest that iPS-like cells can be derived from prostatic epithelial cells. These cells are pluripotent and capable of prostatic differentiation, and therefore provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification. Bisulphite converted DNA from the 10 samples were hybridised to the Illumina Infinium HumanMethylation450K Beadchip v1.2

Dataset Information

Cell Fusion Enhances Mesendodermal Differentiation of Human Induced Pluripotent Stem Cells.

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Cell fusion enhances mesendodermal differentiation of human induced pluripotent stem cells.

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