Multipotent Cell Lineage Research Articles

The transcriptomic landscape of caudal cell mass in different developmental stages of the chick embryo.

The caudal cell mass (CCM) is an aggregate of undifferentiated pluripotent cells and the main player in secondary neurulation. Previous studies have elucidated the dynamic fate of the multipotent cell lineages, with arecent interest in the neuromesodermal progenitors. However, a transcriptomic analysis of the CCM during secondary neurulation has not been performed yet. We analyzed RNA sequencing data of CCM samples at three different developmental stages of chicken embryos; HH16 (largest CCM phase), HH20 (secondary neural tube formation phase), and HH28 (degeneration phase). The transcriptomic profiles were clearly distinguishable according to developmental stage, and HH20 was shown to have not only intermediate, but also unique properties in secondary neurulation. A total of 10,666 differentially expressed genes, including FGF18 and GDF11, were identified and enriched in several gene ontologies related to embryogenesis or organogenesis. We also found that genes encoding transcription factors, such as TWIST2, IRX4, HOXB4, HOXD13, LIN28A, CDX4, and Brachyury, were among the top-ranked differentially expressed genes. Through transcriptomic profiling, we provided a picture of the developmental process of the CCM. We identified several key molecules or pathways involved in secondary neurulation and the pathogenesis of related diseases.

ID:3004 Lung cancer stem cell and its therapeutic potential

Cancer stem cells (CSCs), a subpopulation of cancer cells with the ability to self-renew and giving rise to differentiated cancer cells, are thought to play a crucial role in tumour development, invasion, metastasis and recurrence. Treatments for lung cancer remain a challenging due to ineffectiveness of current available therapies to eradicate the quiescent and therapy-resistance of CSCs. Therefore, new treatment regime specifically targeting CSCs are needed to improve the effectiveness of lung cancer treatment. However the identification of lung CSCs especially in non-small cell lung cancer (NSCLC) remains challenging due to highly heterogeneous cell population and no standard markers to distinguish the lung CSCs from their cancer origins. The use of cell surface markers in isolation, identification and characterization of CSCs is one of the important tools as to understand the basic molecular composition and properties of CSCs and the pathways that of these heterogeneous population of cells are involved for future targeted therapy. Our group has identified, isolated and characterized putative lung CSCs from NSCLC cell line based on expression of combination CSCs markers, the CD166/CD44 and CD166/EpCAM using fluorescence activated cell sorting (FACS). The putative lung CSCs, the CD166+CD44+ CD166+EpCAM+ were characterized for multipotent and self-renewal capability using differentiation, clonogenic and spheroid assays. Following microarray gene expression study and bioinformatics analysis, our data has shown that the NF-κB was consistently expressed and shown to play a significant role in maintaining tumorigenicity of CSCs. In order to proof the role of NF-κB, the cells were treated with different concentrations of BMS-345541 (an inhibitor of NF-κB molecule) and the effectiveness of the treatments were evaluated through its ability to reduce self-renewal, expression of stemness genes (Sox2, Oct4, Klf4, Nanog and Sca-1), migration and epithelial to mesenchymal transition process (EMT) activities. The effectiveness of curcumin as drug sensitizer for effective therapy in treating chemoresistance of CSCs, was also investigated. The capability of CD166+/CD44+ and CD166+/EpCAM+ to differentiate into multipotent cell lineages (adipocytes and osteocyte), to form colony and to anchorage independent spheres and expressed stemness genes were shown in this study. The inhibition of NF-κB molecule using BMS-345441 has reduced the sphere forming capability, expression of stemness genes (Sox2, Nanog and Oct4), migration and expression of EMT genes in lung CSCs. Meanwhile, curcumin was found to reduce the cells proliferation activity and induced apoptosis of the cells. In addition, curcumin was also able to sensitize cisplatin, to supress the migration ability and inhibiting self-renewal capability of lung CSCs. Besides, the mRNA level of Sox2, Klf4, Nanog and Oct4 were down-regulated in lung CSCs after treatment with combination of both curcumin and cisplatin. Our findings suggest that NF-κB could be used as a potential targeted gene for lung cancer gene modification whereas curcumin could be used as potential natural compound that was able to sensitize the chemotherapy drug for effective cancer therapy.

Molecular Reproduction & Development Volume 82, Issue 6, June 2015 Cover Image

A 10‐day old sea urchin larva (Strongylocentrotus purpuratus) expands the left coelomic pouch (red) to form future adult precursor tissues, including the germ line. A germ‐line marker, Vasa (red), which is initially expressed in every blastomere of the sea urchin during early embryogenesis (see Yajima and Wessel, this issue), is retained in multipotent cell lineages during late larval development (DNA, cyan). This image shows a lateral view from the left side of the larva.

Hyaluronan-CD44v3 Interaction with Oct4-Sox2-Nanog Promotes miR-302 Expression Leading to Self-renewal, Clonal Formation, and Cisplatin Resistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

The metalloproteinase inhibitor Reck is essential for zebrafish DRG development

The neural crest is a migratory, multipotent cell lineage that contributes to myriad tissues, including sensory neurons and glia of the dorsal root ganglia (DRG). To identify genes affecting cell fate specification in neural crest, we performed a forward genetic screen for mutations causing DRG deficiencies in zebrafish. This screen yielded a mutant lacking all DRG, which we named sensory deprived (sdp). We identified a total of four alleles of sdp, all of which possess lesions in the gene coding for reversion-inducing cysteine-rich protein containing Kazal motifs (Reck). Reck is an inhibitor of metalloproteinases previously shown to regulate cell motility. We found reck function to be both necessary for DRG formation and sufficient to rescue the sdp phenotype. reck is expressed in neural crest cells and is required in a cell-autonomous fashion for appropriate sensory neuron formation. In the absence of reck function, sensory neuron precursors fail to migrate to the position of the DRG, suggesting that this molecule is crucial for proper migration and differentiation.

SATB Homeobox Proteins Regulate Trophoblast Stem Cell Renewal and Differentiation

The morphogenesis of the hemochorial placenta is dependent upon the precise expansion and differentiation of trophoblast stem (TS) cells. SATB homeobox 1 (SATB1) and SATB2 are related proteins that have been implicated as regulators of some stem cell populations. SATB1 is highly expressed in TS cells, which prompted an investigation of SATB1 and the related SATB2 as regulators of TS cells. SATB1 and SATB2 were highly expressed in rat TS cells maintained in the stem state and rapidly declined following induction of differentiation. SATB proteins were also present within the rat placenta during early stages of its morphogenesis and disappeared as gestation advanced. Silencing Satb1 or Satb2 expression decreased TS cell self-renewal and increased differentiation, whereas ectopic expression of SATB proteins promoted TS cell expansion and blunted differentiation. Eomes, a key transcriptional regulator of TS cells, was identified as a target for SATB proteins. SATB knockdown decreased Eomes transcript levels and promoter activity, whereas SATB ectopic expression increased Eomes transcript levels and promoter activity. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation analyses demonstrated that SATB proteins physically associate with a regulatory site within the Eomes promoter. We conclude that SATB proteins promote TS cell renewal and inhibit differentiation. These actions are mediated in part by regulating the expression of the TS cell stem-associated transcription factor, EOMES.

Large Scale Phosphoproteome Profiles Comprehensive Features of Mouse Embryonic Stem Cells

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Cytokines Instruct Hematopoietic Lineage Choice

AbstractAbstract SCI-13The molecular control of multipotent cell lineage choice is one of the central questions in stem cell biology. Since decades, it is controversial whether cell extrinsic factors can actively influence lineage decisions of hematopoietic cells (instructive model). Alternatively, they may only allow the survival and proliferation of already unilineage-restricted cells, which have chosen one lineage by cell-autonomous mechanisms (selective / permissive model). One major reason for this longstanding controversy is the fact that hematopoiesis is usually followed by analyzing populations of cells - rather than individual cells - at very few time points of an experiment and without knowing their individual identities. However, the alternative lineage choice models can only be distinguished by constantly observing individual cell fates throughout differentiation.We therefore applied novel computer aided culture, imaging and cell tracking approaches to follow the fate of individual cells over many days. Analysis of primary mouse multipotent hematopoietic cell differentiation finally provided proof for the lineage instructive action of hematopoietic cytokines. This finding is of fundamental importance for our understanding of the molecular control of lineage choice. It shows that cell extrinsic signaling influences the - currently unknown - cell intrinsic molecular lineage choice and commitment machinery. Our novel approaches now allow identification of the signaling pathways mediating lineage instruction and their targets with the required precision. Moreover, we are developing technology for the quantification of lineage specific transcription factor proteins in individual living cells. This novel kind of continuous, live and quantitative molecular and cellular data is used for modeling the control of cell fates through integration of cell extrinsic signals with cell intrinsic molecular states. Disclosures:No relevant conflicts of interest to declare.

Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain.

In many species, the Sox2 transcription factor is a marker of the nervous system from the beginning of its development, and we have previously shown that Sox2 is expressed in embryonic neural stem cells. It is also expressed in, and is essential for, totipotent inner cell mass stem cells and other multipotent cell lineages, and its ablation causes early embryonic lethality. To investigate the role of Sox2 in the nervous system, we generated different mouse mutant alleles: a null allele (Sox2beta-geo 'knock-in'), and a regulatory mutant allele (Sox2DeltaENH), in which a neural cell-specific enhancer is deleted. Sox2 is expressed in embryonic early neural precursors of the ventricular zone and, in the adult, in ependyma (a descendant of the ventricular zone). It is also expressed in the vast majority of dividing precursors in the neurogenic regions, and in a small proportion of differentiated neurones, particularly in the thalamus, striatum and septum. Compound Sox2(beta-geo/DeltaENH) heterozygotes show important cerebral malformations, with parenchymal loss and ventricle enlargement, and L-dopa-rescuable circling behaviour and epilepsy. We observed striking abnormalities in neurones; degeneration and cytoplasmic protein aggregates, a feature common to diverse human neurodegenerative diseases, are observed in thalamus, striatum and septum. Furthermore, ependymal cells show ciliary loss and pathological lipid inclusions. Finally, precursor cell proliferation and the generation of new neurones in adult neurogenic regions are greatly decreased, and GFAP/nestin-positive hippocampal cells, which include the earliest neurogenic precursors, are strikingly diminished. These findings highlight a crucial and unexpected role for Sox2 in the maintenance of neurones in selected brain areas, and suggest a contribution of neural cell proliferative defects to the pathological phenotype.

Multipotent cell lineages in early mouse development depend on SOX2 function.

Each cell lineage specified in the preimplantation mammalian embryo depends on intrinsic factors for its development, but there is also mutual interdependence between them. OCT4 is required for the ICM/epiblast lineage, and at transient high levels for extraembryonic endoderm, but also indirectly through its role in regulating Fgf4 expression, for the establishment and proliferation of extraembryonic ectoderm from polar trophectoderm. The transcription factor SOX2 has also been implicated in the regulation of Fgf4 expression. We have used gene targeting to inactivate Sox2, examining the phenotypic consequences in mutant embryos and in chimeras in which the epiblast is rescued with wild-type ES cells. We find a cell-autonomous requirement for the gene in both epiblast and extraembryonic ectoderm, the multipotent precursors of all embryonic and trophoblast cell types, respectively. However, an earlier role within the ICM may be masked by the persistence of maternal protein, whereas the lack of SOX2 only becomes critical in the chorion after 7.5 days postcoitum. Our data suggest that maternal components could be involved in establishing early cell fate decisions and that a combinatorial code, requiring SOX2 and OCT4, specifies the first three lineages present at implantation.