Pluripotent Precursor Cells Research Articles

Glycosylation as a tracer of off-target Cre-lox activation in development.

The Cre-lox system is one of the most widely used methods for lineage-specific and inducible genome editing in vivo. However, incomplete penetrance and off-target effects due to transient promoter expression in a stem or pluripotent precursor cell can be problematic and difficult to detect, especially if the target gene is not normally present in the fully differentiated but off-target cells. Yet, the loss of the target gene through the transient expression of Cre may impact the differentiation of those cells by virtue of transient expression in a precursor population. In these situations, off-target effects in an unknown precursor cell can, at best, complicate conclusions drawn from the model, and at worst, invalidate all data generated from that knockout strain. Thus, identifying Cre-driver promoter expression along entire cell lineages is crucial to improve rigor and reproducibility. As an example, transient expression in an early precursor cell has been documented in a variety of Cre strains such as the Tie2-based Cre-driver system that is used as an "endothelial cell-specific" model 1. Yet, Tie2 is now known to be transiently expressed in a stem cell upstream of both hematopoietic and endothelial cell lineages. Here, we use the Tie2 Cre-driver strain to demonstrate that due to its ubiquitous nature, plasma membrane glycans are a useful marker of both penetrance and specificity of a Cre-based knockout.

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.

The plant stem-cell niche and pluripotency: 15 years of an epigenetic perspective.

Pluripotent stem-cells are slowly dividing cells giving rise to daughter cells that can either differentiate to new tissues and organs, or remain stem-cells. In plants, stem-cells are located in specific niches of the shoot and root apical meristems (SAMs and RAMs). After ablation of stem-cell niches, pluripotent meristematic cells can establish new stem-cells, whereas the removal of the whole meristem destructs the regeneration process. In tissue cultures, after detached plant organs are transferred to rooting or callus induction medium (G5 or CIM), vasculature-associated pluripotent cells (VPCs) immediately start proliferation to form adventitious roots or callus, respectively, while other cell types of the organ explants basically play no part in the process. Hence, in contrast to the widely-held assumption that all plant cells have the ability to reproduce a complete organism, only few cell types are pluripotent in practice, raising the question how pluripotent stem-cells differ from differentiated cells. It is now clear that, in addition to gene regulatory networks of pluripotency factors and phytohormone signaling, epigenetics play a crucial role in initiation, maintenance and determination of plant stem-cells. Although, more and more epigenetic regulators have been shown to control plant stem-cell fate, only a few studies demonstrate how they are recruited and how they change the chromatin structure and transcriptional regulation of pluripotency factors. Here, we highlight recent breakthroughs but also revisited classical studies of epigenetic regulation and chromatin dynamics of plant stem-cells and their pluripotent precursor-cells, and point out open questions and future directions.

Genomic and transcriptomic profiling of hepatoid adenocarcinoma of the stomach.

Hepatoid adenocarcinoma of the stomach (HAS), a rare subtype of gastric cancer (GC), has a low incidence but a high mortality rate. Little is known about the molecular features of HAS. Here we applied whole-exome sequencing (WES) on 58 tumours and the matched normal controls from 54 HAS patients, transcriptome sequencing on 30 HAS tumours, and single-cell RNA sequencing (scRNA-seq) on one HAS tumour. Our results reveal that the adenocarcinomatous component and hepatocellular-like component of the same HAS tumour originate monoclonally, and HAS is likely to initiate from pluripotent precursor cells. HAS has high stemness and high methionine cycle activity compared to classical GC. Two genes in the methionine cycle, MAT2A, and AHCY are potential targets for HAS treatments. We provide the first integrative genomic profiles of HAS, which may facilitate its diagnosis, prognosis, and treatment.

IL-10-Producing Potential Treg Precursor in Placenta.

The maternal immune system needs to be tolerant of allogeneic fetal tissue for reproductive success. The regulatory immune cell network plays an essential role in maintaining maternal tolerance to the fetus. We herein demonstrate in a green fluorescent protein (GFP)/IL-10 reporter mouse system that unique IL-10-expressing cells exist presumably in chorionic villi within the placenta. Flow cytometric analysis revealed that these IL-10- expressing cells exhibit a unique CD19 negative, CD3 negative, and B220 positive phenotype. Interestingly, these cells were enriched during in vitro culture, but well-known stimuli for T cells and B cells failed to enhance their growth, suggesting that the CD19- CD3- B220+ cells were self renewing. Unexpectedly, in an adoptive cell trans fer experiment, IL-10 production was detected in Sca-1+ CD4+ CD25+ regulatory T cells (Treg). To our knowledge, this is the first report to identify IL-10-producing CD19- CD3- B220+ cells in the fetus. These cells may rep resent a potential progenitor of Sca-1+ Treg or pluripotent precursor cells for immune tolerance.

Defining Epigenetic Regulation of the Interleukin-9 Gene by Chromatin Immunoprecipitation.

Regulation of gene expression is essential for the differentiation of pluripotent precursor cells into specialized effector cells and, thus, for the evolution of multiorgan systems. The regulation of gene expression is controlled by a variety of "extra-genic" mechanisms, termed as epigenetic mechanisms. Obviously, alterations in such control mechanisms of gene expression may result in alterations of cellular effector functions, resulting in for example defects in cellular functions, but also, if immune cells are involved, leading to the development of immunologic disorders such as malignancies and autoimmune diseases. The analysis of epigenetic modifications is therefore pertinent not only for the understanding of the regular function of the immune system, but also for the understanding of the pathophysiology of such diseases. As interleukin-9 (IL-9) is the signature cytokine for Th9 cells, and since IL-9 plays important roles in the orchestration of a protective immune responses, the analysis of epigenetic mechanisms underlying the development of IL-9 producing effector T cells is of great interest. Here, we describe a protocol to analyze epigenetic regulation of the IL-9 gene by chromatin immunoprecipitation.

Direct conversion of fibroblasts traces the way back to our first organ-the placenta.

Cell fate determination has always fascinated scientists predominantly embryologists and challenged them to decipher master genes or networks of transcriptional factors which together with cell signaling cascades act as key regulators for the cell lineage allocation. With the rediscovery of the pluripotent precursor cells in an adult body and the possibility to induce pluripotency in somatic cells (iPSCs), as sources for alternative therapeutic options in translational medicine, this field has attracted high interest in medical research and application (1). iPSC have several advantages over embryonic stem cells such as avoiding tumor formation and to circumvent ethical concerns. Meanwhile the understanding of pluripotency has broadened thanks to abundant studies on converting ESC and somatic cells into another cell fate using predominantly transfection of a bunch of lineage specific transcription factors. While many studies focused on the regulation of ESC conversion less is known about the hierarchically organized network of transcriptional factors which determine the trophoblast lineage for placenta development.

Molecular Characteristics of Malignant Ovarian Germ Cell Tumors and Comparison With Testicular Counterparts: Implications for Pathogenesis

This review focuses on the molecular characteristics and development of rare malignant ovarian germ cell tumors (mOGCTs). We provide an overview of the genomic aberrations assessed by ploidy, cytogenetic banding, and comparative genomic hybridization. We summarize and discuss the transcriptome profiles of mRNA and microRNA (miRNA), and biomarkers (DNA methylation, gene mutation, individual protein expression) for each mOGCT histological subtype. Parallels between the origin of mOGCT and their male counterpart testicular GCT (TGCT) are discussed from the perspective of germ cell development, endocrinological influences, and pathogenesis, as is the GCT origin in patients with disorders of sex development. Integrated molecular profiles of the 3 main histological subtypes, dysgerminoma (DG), yolk sac tumor (YST), and immature teratoma (IT), are presented. DGs show genomic aberrations comparable to TGCT. In contrast, the genome profiles of YST and IT are different both from each other and from DG/TGCT. Differences between DG and YST are underlined by their miRNA/mRNA expression patterns, suggesting preferential involvement of the WNT/β-catenin and TGF-β/bone morphogenetic protein signaling pathways among YSTs. Characteristic protein expression patterns are observed in DG, YST and IT. We propose that mOGCT develop through different developmental pathways, including one that is likely shared with TGCT and involves insufficient sexual differentiation of the germ cell niche. The molecular features of the mOGCTs underline their similarity to pluripotent precursor cells (primordial germ cells, PGCs) and other stem cells. This similarity combined with the process of ovary development, explain why mOGCTs present so early in life, and with greater histological complexity, than most somatic solid tumors.

Mouse stem cells seeded into decellularized rat kidney scaffolds endothelialize and remodel basement membranes

Introduction To address transplant organ shortage, a promising strategy is to decellularize kidneys in a manner that the scaffold retains signals for seeded pluripotent precursor cells to differentiate and recapitulate native structures: matrix-to-cell signaling followed by cell-cell and cell-matrix interactions, thereby remodeling and replacing the original matrix. This would reduce scaffold antigenicity and enable xeno-allografts.Results DAPI-labeled cells in arterial vessels and glomeruli were positive for both endothelial lineage markers, BsLB4 and VEGFR2. Rat scaffold’s basement membrane demonstrated immunolabeling with anti-mouse laminin β1. Labeling intensified over time with 14 day incubations.Conclusion We provide new evidence for matrix-to-cell signaling in acellular whole organ scaffolds that induces differentiation of pluripotent precursor cells to endothelial lineage. Production of mouse basement membrane supports remodeling of host (rat)-derived scaffolds and thereby warrants further investigation as a promising approach for xenotransplantation.Methods We previously showed that murine embryonic stem cells arterially seeded into acellular rat whole kidney scaffolds multiply and demonstrate morphologic, immunohistochemical and gene expression evidence for differentiation. Vascular cell endothelialization was now further tested by endothelial specific BsLB4 lectin and anti-VEGFR2 (Flk1) antibodies. Remodeling of the matrix basement membranes from rat to mouse (“murinization”) was assessed by a monoclonal antibody specific for mouse laminin β1 chain.

Squelching of ETS2 Transactivation by POU5F1 Silences the Human Chorionic Gonadotropin CGA Subunit Gene in Human Choriocarcinoma and Embryonic Stem Cells

The subunit genes encoding human chorionic gonadotropin, CGA, and CGB, are up-regulated in human trophoblast. However, they are effectively silenced in choriocarcinoma cells by ectopically expressed POU domain class 5 transcription factor 1 (POU5F1). Here we show that POU5F1 represses activity of the CGA promoter through its interactions with ETS2, a transcription factor required for both placental development and human chorionic gonadotropin subunit gene expression, by forming a complex that precludes ETS2 from interacting with the CGA promoter. Mutation of a POU5F1 binding site proximal to the ETS2 binding site does not alter the ability of POU5F1 to act as a repressor but causes a drop in basal promoter activity due to overlap with the binding site for DLX3. DLX3 has only a modest ability to raise basal CGA promoter activity, but its coexpression with ETS2 can up-regulate it 100-fold or more. The two factors form a complex, and both must bind to the promoter for the combination to be transcriptionally effective, a synergy compromised by POU5F1. Similarly, in human embryonic stem cells, which express ETS2 but not CGA, ETS2 does not occupy its binding site on the CGA promoter but is found instead as a soluble complex with POU5F1. When human embryonic stem cells differentiate in response to bone morphogenetic protein-4 and concentrations of POU5F1 fall and hCG and DLX3 rise, ETS2 then occupies its binding site on the CGA promoter. Hence, a squelching mechanism underpins the transcriptional silencing of CGA by POU5F1 and could have general relevance to how pluripotency is maintained and how the trophoblast lineage emerges from pluripotent precursor cells.

The Synergistic Effects of Agarose Scaffold Supplemented with Low-molecular-weight Silk Fibroin in Bone Tissue Regeneration

Silk protein and agarose are widely known as biocompatible materials in the human body. A three-dimensional (3D) scaffold composed of agarose and low-molecular- weight silk fibroin (LSF) was fabricated and examined in terms of structural characteristics and cellular responses in bone tissue engineering. This study showed that mouse pluripotent precursor cells attached to and proliferated uniformly on and within the LSF-containing 3D scaffold. Interestingly, cell proliferation and attachment was shown to be higher in a 3D scaffold containing 0.02% LSF, as compared to other LSF concentrations. The results of this study suggest that agarose-LSF scaffolds may be useful materials for tissue engineering.

The BTB-ZF Family of Transcription Factors: Key Regulators of Lineage Commitment and Effector Function Development in the Immune System

Successful immunity depends upon the activity of multiple cell types. Commitment of pluripotent precursor cells to specific lineages, such as T or B cells, is obviously fundamental to this process. However, it is also becoming clear that continued differentiation and specialization of lymphoid cells is equally important for immune system integrity. Several members of the BTB-ZF family have emerged as critical factors that control development of specific lineages and also of specific effector subsets within these lineages. For example, BTB-ZF genes have been shown to control T cell versus B cell commitment and CD4 versus CD8 lineage commitment. Others, such as PLZF for NKT cells and Bcl-6 for T follicular helper cells, are necessary for the acquisition of effector functions. In this review, we summarize current findings concerning the BTB-ZF family members with a reported role in the immune system.

Retinoic acid-induced Smad3 expression is required for the induction of osteoblastogenesis of mesenchymal stem cells

Mesenchymal stem cells are pluripotent precursor cells that can differentiate into osteoblasts, adipocytes, chondrocytes and myocytes. Despite their important therapeutic potential little is known about the transcriptional cascades that govern lineage decisions in these cells. Treatment of C3H10T1/2 mouse mesenchymal stem cells with retinoic acid (RA) inhibits adipogenesis and enhances osteoblastogenesis. In particular, RA treatment stimulates the expression of the osteoblast master regulator, runt-related transcription factor 2 (Runx2), whose expression is necessary for the formation of bone. We have shown previously in mesenchymal stem cells that RA acts to stimulate osteoblastogenesis by interfering with the actions of the bzip transcription factor CCAAT/Enhancer Binding Protein beta (C/EBPβ), where it binds to a negative regulatory element within the Runx2 promoter and inhibits its expression. Herein we show that Smad3, whose expression is stimulated by RA, relays the effects of RA on differentiation by initiating the displacement of C/EBPβ from the Runx2 promoter. In addition to stimulating Smad3 expression, RA also stimulated the nuclear localization of this factor, such that in the absence of RA, ectopic Smad3 was unable to drive osteoblastogenesis. While not sufficient to promote osteoblastogenesis, knockdown of Smad3 using a specific shRNA prevented the RA-mediated stimulation of differentiation and displacement of C/EBPβ from the Runx2 P1 promoter. Taken together, these data indicate that Smad3 is an important mediator of RA activity during mesenchymal stem cell differentiation and is necessary for the stimulation of osteoblastogenesis.

Influence of Altered Local Effective Concentration on Cytoskeletal and Ecm Structure in Human Mesenchymal Stem Cells

Mesenchymal stromal or stem cells (MSCs) are a type of pluripotent precursor cell found in bone marrow, which can be coaxed in vitro to express certain characteristics of differentiated mesenchymal tissue lineages. Many therapeutic applications of MSCs require isolation from a given patient, expansion to useful cell numbers in culture, and reimplantation. However, the in vivo and in vitro culture conditions remain in stark contrast, and confound interpretation of experiments and development of therapeutic uses for MSCs. For example, in vivo, the extracellular microenvironment is highly crowded with proteins; in vitro, the extracellular culture media is decidedly dilute. Here, we use multiple methods to consider whether this differential crowding can directly alter MSC structure and function. We consider a series of macromolecular crowders within the culture media of MSCs, expanded under basal (non-differentiating) conditions. We then quantify changes in population-level proliferation and migration, and in extracellular and intracellular protein structure and cortical cytoskeletal stiffness via immunocytochemistry, optical microscopy, and atomic force microscopy-enabled imaging and nanoindentation of living MSCs. We find significant effects of “crowded” culture conditions on cell architecture, extracellular matrix structure, and attendant cell properties. These results motivate the development of more physiologically relevant environments for both in vitro studies and biodevices, to ultimately answer fundamental questions of physiological and pathological behavior for MSCs.

Programming pluripotent precursor cells derived from Xenopus embryos to generate specific tissues and organs.

Xenopus embryos provide a rich source of pluripotent cells that can be differentiated into functional organs. Since the molecular principles of vertebrate organogenesis appear to be conserved between Xenopus and mammals, this system can provide useful guidelines for the directional manipulation of human embryonic stem cells. Pluripotent Xenopus cells can be easily isolated from the animal pole of blastula stage Xenopus embryos. These so called “animal cap” cells represent prospective ectodermal cells, but give rise to endodermal, mesodermal and neuro-ectodermal derivatives if treated with the appropriate factors. These factors include evolutionary conserved modulators of the key developmental signal transduction pathways that can be supplied either by mRNA microinjection or direct application of recombinant proteins. This relatively simple system has added to our understanding of pancreas, liver, kidney, eye and heart development. In particular, recent studies have used animal cap cells to generate ectopic eyes and hearts, setting the stage for future work aimed at programming pluripotent cells for regenerative medicine.

Hematopoietic stem cells in Drosophila

The Drosophila lymph gland, the source of adult hemocytes, is established by mid-embryogenesis. During larval stages, a pool of pluripotent hemocyte precursors differentiate into hemocytes that are released into circulation upon metamorphosis or in response to immune challenge. This process is controlled by the posterior signaling center (PSC), which is reminiscent of the vertebrate hematopoietic stem cell niche. Using lineage analysis, we identified bona fide hematopoietic stem cells (HSCs) in the lymph glands of embryos and young larvae, which give rise to a hematopoietic lineage. These lymph glands also contain pluripotent precursor cells that undergo a limited number of mitotic divisions and differentiate. We further find that the conserved factor Zfrp8/PDCD2 is essential for the maintenance of the HSCs, but dispensable for their daughter cells, the pluripotent precursors. Zfrp8/PDCD2 is likely to have similar functions in hematopoietic stem cell maintenance in vertebrates.

Cellular and molecular mechanisms responsible for the action of testosterone on human skeletal muscle. A basis for illegal performance enhancement

The popularity of testosterone among drug users is due to its powerful effects on muscle strength and mass. Important mechanisms behind the myotrophic effects of testosterone were uncovered both in athletes using steroids for several years and in short-term controlled studies. Both long-term and short-term steroid usage accentuates the degree of fibre hypertrophy in human skeletal muscle by enhancing protein synthesis. A mechanism by which testosterone facilitates the hypertrophy of muscle fibres is the activation of satellite cells and the promotion of myonuclear accretion when existing myonuclei become unable to sustain further enhancement of protein synthesis. Interestingly, long-term steroid usage also enhances the frequency of fibres with centrally located myonuclei, which implies the occurrence of a high regenerative activity. Under the action of testosterone, some daughter cells generated by satellite cell proliferation may escape differentiation and return to quiescence, which help to replenish the satellite cell reserve pool. However, whether long-term steroid usage induces adverse effects of satellite cells remains unknown. Testosterone might also favour the commitment of pluripotent precursor cells into myotubes and inhibit adipogenic differentiation. The effects of testosterone on skeletal muscle are thought to be mediated via androgen receptors expressed in myonuclei and satellite cells. Some evidence also suggests the existence of an androgen-receptor-independent pathway. Clearly, testosterone abuse is associated with an intense recruitment of multiple myogenic pathways. This provides an unfair advantage over non-drug users. The long-term consequences on the regenerative capacity of skeletal muscle are unknown.

Methacholine and PDGF activate store-operated calcium entry in neuronal precursor cells via distinct calcium entry channels

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signals that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signals involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of release from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.

Composite signet-ring cell/neuroendocrine carcinoma of the stomach with a metastatic neuroendocrine carcinoma component: a better prognosis entity

BackgroundMixed (composite) exocrine-neuroendocrine cell carcinomas are defined as an intimate admixture of neoplastic glandular exocrine and neuroendocrine cell types. Although gastric adenocarcinoma containing a small number of neuroendocrine cells is a relatively frequent occurrence, gastric neoplasms containing equal proportions of both cell types are rare.Case PresentationWe present a case of composite exocrine (signet-ring cell)-neuroendocrine cell carcinoma, in which the neoplastic signet-ring cell exocrine and neuroendocrine constituents occurred in fairly equivalent amounts, whereas only the neuroendocrine carcinoma portion of the tumor represented the metastatic component. Light microscopy, immunohistochemical and electron microscopic findings are described, and the literature is reviewed.ConclusionThis study confirms the ability of pluripotent precursor cells to differentiate into either adenocarcinoma or neuroendocrine tumor and, justifying the designation of composite exocrine-neuroendocrine cell carcinoma as the appropriate classification for this tumor. The protracted clinical course further supports the notion that composite signet-ring cell/neuroendocrine carcinoma tumors behave relatively less aggressively than the pure forms of the former cell type.

Role of T-box gene tbx-2 for anterior foregut muscle development in C. elegans

During organogenesis, pluripotent precursor cells acquire a defined identity such as muscle or nerve. The transition from naïve precursor towards the differentiated state is characterized by sequential waves of gene expression that are determined by regulatory transcription factors. A key question is how transcriptional circuitry dictates the succession of events that accompanies developmental competence, cell fate specification and differentiation. To address this question, we have examined how anterior muscles are established within the Caenorhabditis elegans foregut (pharynx). We find that the T-box transcription factor tbx-2 is essential to form anterior pharyngeal muscles from the ABa blastomere. In the absence of tbx-2 function, ABa-derived cells initiate development normally: they receive glp-1/Notch signaling cues, activate the T-box gene TBX-38 and express the organ selector gene PHA-4/FoxA. However, these cells subsequently arrest development, extinguish PHA-4 and fail to activate PHA-4 target genes. tbx-2 mutant cells do not undergo apoptosis and there is no evidence for adoption of an alternative fate. TBX-2 is expressed in ABa descendants and depends on activation by pha-4 and repression by components of glp-1/Notch signaling. Our analysis suggests that a positive feedback loop between tbx-2 and pha-4 is required for ABa-derived precursors to commit to pharyngeal muscle fate.