scholarly journals Comparison of the 3D organization of sperm and fibroblast genomes using the Hi-C approach

2014 ◽  
Author(s):  
Nariman Battulin ◽  
Veniamin S Fishman ◽  
Alexander M Mazur ◽  
Mikhail Pomaznoy ◽  
Anna A Khabarova ◽  
...  
Keyword(s):  
Es Cells ◽  
3D Structure ◽  
Embryonic Stem ◽  
Cell Types ◽  
Chromatin Interaction ◽  
Sperm Cells ◽  
3D Genome ◽  
Genome Wide ◽  
Topologically Associated Domains ◽  
The Stability

The 3D organization of the genome is tightly connected to its biological function. The Hi-C approach was recently introduced as a method that can be used to identify higher-order chromatin interactions genome-wide. The aim of this study was to determine genome-wide chromatin interaction frequencies using the Hi-C approach in mouse sperm cells and embryonic fibroblasts. The obtained results demonstrated that the 3D genome organizations of sperm and fibroblast cells show a high degree of similarity both with each other and with the previously described mouse embryonic stem (ES) cells. Both A- and B-compartments and topologically associated domains (TADs) are present in spermatozoa and fibroblasts. Nevertheless, sperm cells and fibroblasts exhibited statistically significant differences between each other in the contact probabilities of defined loci. Tight packaging of the sperm genome resulted in an enrichment of long-range contacts compared with the fibroblasts. However, only 30% of the differences in the number of contacts are based on differences in the densities of their genome packages; the main source of the differences is the gain or loss of contacts that are specific for defined genome regions. An analysis of interchromosomal contacts in both cell types demonstrated that the large chromosomes showed a tendency to interact with each other more than with the small chromosomes and vice versa. We found that the dependence of the contact probability P(s) on genomic distance for sperm is in a good agreement with the fractal globular folding of chromatin. The similarity of the spatial DNA organization in sperm and somatic cell genomes suggests the stability of the 3D structure of genomes through generations.

scholarly journals Epigenetic Regulation of Mesenchymal Stem Cells: A Focus on Osteogenic and Adipogenic Differentiation

2011 ◽  
Vol 2011 ◽  
pp. 1-18 ◽  
Author(s):  
Chad M. Teven ◽  
Xing Liu ◽  
Ning Hu ◽  
Ni Tang ◽  
Stephanie H. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Epigenetic Regulation ◽  
Adult Stem Cells ◽  
Es Cells ◽  
Adipogenic Differentiation ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiation Potential ◽  
Msc Differentiation

Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES) cells. Mesenchymal stem cells (MSCs) are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.

scholarly journals Mast Cell-Specific Expression of Human Siglec-8 in Conditional Knock-in Mice

2018 ◽  
Vol 20 (1) ◽  
pp. 19 ◽  
Author(s):  
Yadong Wei ◽  
Krishan Chhiba ◽  
Fengrui Zhang ◽  
Xujun Ye ◽  
Lihui Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mast Cell ◽  
Mast Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Surface Protein ◽  
Cell Types ◽  
Mouse Strains ◽  
Specific Expression

Sialic acid-binding Ig-like lectin 8 (Siglec-8) is expressed on the surface of human eosinophils, mast cells, and basophils—cells that participate in allergic and other diseases. Ligation of Siglec-8 by specific glycan ligands or antibodies triggers eosinophil death and inhibits mast cell degranulation; consequences that could be leveraged as treatment. However, Siglec-8 is not expressed in murine and most other species, thus limiting preclinical studies in vivo. Based on a ROSA26 knock-in vector, a construct was generated that contains the CAG promoter, a LoxP-floxed-Neo-STOP fragment, and full-length Siglec-8 cDNA. Through homologous recombination, this Siglec-8 construct was targeted into the mouse genome of C57BL/6 embryonic stem (ES) cells, and chimeric mice carrying the ROSA26-Siglec-8 gene were generated. After cross-breeding to mast cell-selective Cre-recombinase transgenic lines (CPA3-Cre, and Mcpt5-Cre), the expression of Siglec-8 in different cell types was determined by RT-PCR and flow cytometry. Peritoneal mast cells (dual FcεRI+ and c-Kit+) showed the strongest levels of surface Siglec-8 expression by multicolor flow cytometry compared to expression levels on tissue-derived mast cells. Siglec-8 was seen on a small percentage of peritoneal basophils, but not other leukocytes from CPA3-Siglec-8 mice. Siglec-8 mRNA and surface protein were also detected on bone marrow-derived mast cells. Transgenic expression of Siglec-8 in mice did not affect endogenous numbers of mast cells when quantified from multiple tissues. Thus, we generated two novel mouse strains, in which human Siglec-8 is selectively expressed on mast cells. These mice may enable the study of Siglec-8 biology in mast cells and its therapeutic targeting in vivo.

scholarly journals Designer blood: creating hematopoietic lineages from embryonic stem cells

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1265-1275 ◽  
Author(s):  
Abby L. Olsen ◽  
David L. Stachura ◽  
Mitchell J. Weiss
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Basic Research ◽  
Cell Types ◽  
Patient Specific ◽  
Es Cell ◽  
Blood Disorders ◽  
Hematopoietic Stem

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.

scholarly journals Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

2019 ◽  
Author(s):  
Aseda Tena ◽  
Yuxiang Zhang ◽  
Nia Kyritsis ◽  
Anne Devorak ◽  
Jeffrey Zurita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Progenitors ◽  
Neural Genes ◽  
Genome Wide ◽  
Primary Mouse

ABSTRACTMild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

scholarly journals Tissue-specific trans regulation of the mouse epigenome

2018 ◽  
Author(s):  
Christopher L. Baker ◽  
Michael Walker ◽  
Seda Arat ◽  
Guruprasad Ananda ◽  
Pavlina Petkova ◽  
...  
Keyword(s):  
Germ Cells ◽  
Es Cells ◽  
Recombinant Inbred Lines ◽  
Embryonic Stem ◽  
Cell Types ◽  
Dna Double Strand Breaks ◽  
Male Germ Cells ◽  
Tissue Specific ◽  
Natural Genetic Variation ◽  
Regulatory Loci

ABSTRACTAlthough a variety of writers, readers, and erasers of epigenetic modifications are known, we have little information about the underlying regulatory systems controlling the establishment and maintenance of the epigenetic landscape, which varies greatly among cell types. Here, we have explored how natural genetic variation impacts the epigenome in mice. Studying levels of H3K4me3, a histone modification at sites such as promoters, enhancers, and recombination hotspots, we found tissue-specific trans-regulation of H3K4me3 levels in four highly diverse cell types: male germ cells, embryonic stem (ES) cells, hepatocytes and cardiomyocytes. To identify the genetic loci involved, we measured H3K4me3 levels in male germ cells in a mapping population of 60 BXD recombinant inbred lines, identifying extensive trans-regulation primarily controlled by six major histone quantitative trait loci (hQTL). These chromatin regulatory loci act dominantly to suppress H3K4me3, which at hotspots reduces the likelihood of subsequent DNA double-strand breaks. QTL locations do not correspond with enzyme known to metabolize chromatin features. Instead their locations match clusters of zinc finger genes, making these possible candidates that explain the dominant suppression of H3K4me3. Collectively, these data describe an extensive, tissue-specific set of chromatin regulatory loci that control functionally related chromatin sites.

scholarly journals Capturing cell type-specific chromatin structural patterns by applying topic modeling to single-cell Hi-C data

2019 ◽  
Author(s):  
Hyeon-Jin Kim ◽  
Galip Gürkan Yardımcı ◽  
Giancarlo Bonora ◽  
Vijay Ramani ◽  
Jie Liu ◽  
...  
Keyword(s):  
Single Cell ◽  
Topic Modeling ◽  
Biological Information ◽  
Chromatin Interaction ◽  
Cell Type ◽  
3D Genome ◽  
Genome Wide ◽  
Significant Barrier ◽  
Chromatin Structural ◽  
Cell Type Specific

AbstractSingle-cell Hi-C (scHi-C) interrogates genome-wide chromatin interaction in individual cells, allowing us to gain insights into 3D genome organization. However, the extremely sparse nature of scHi-C data poses a significant barrier to analysis, limiting our ability to tease out hidden biological information. In this work, we approach this problem by applying topic modeling to scHi-C data. Topic modeling is well-suited for discovering latent topics in a collection of discrete data. For our analysis, we generate twelve different single-cell combinatorial indexed Hi-C (sciHi-C) libraries from five human cell lines (GM12878, H1Esc, HFF, IMR90, and HAP1), consisting over 25,000 cells. We demonstrate that topic modeling is able to successfully capture cell type differences from sciHi-C data in the form of “chromatin topics.” We further show enrichment of particular compartment structures associated with locus pairs in these topics.

Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2339-2348 ◽  
Author(s):  
B. Pain ◽  
M.E. Clark ◽  
M. Shen ◽  
H. Nakazawa ◽  
M. Sakurai ◽  
...  
Keyword(s):  
Culture System ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Telomerase Activity ◽  
Specific Antibodies ◽  
Typical Morphology ◽  
Germinal Tissue

Petitte, J.N., Clarck, M.E., Verrinder Gibbins, A. M. and R. J. Etches (1990; Development 108, 185–189) demonstrated that chicken early blastoderm contains cells able to contribute to both somatic and germinal tissue when injected into a recipient embryo. However, these cells were neither identified nor maintained in vitro. Here, we show that chicken early blastoderm contains cells characterised as putative avian embryonic stem (ES) cells that can be maintained in vitro for long-term culture. These cells exhibit features similar to those of murine ES cells such as typical morphology, strong reactivity toward specific antibodies, cytokine-dependent extended proliferation and high telomerase activity. These cells also present high capacities to differentiate in vitro into various cell types including cells from ectodermic, mesodermic and endodermic lineages. Production of chimeras after injection of the cultivated cells reinforced the view that our culture system maintains in vitro some avian putative ES cells.

Development to term of mouse androgenetic aggregation chimeras

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1325-1333 ◽  
Author(s):  
J.R. Mann ◽  
C.L. Stewart
Keyword(s):  
Mouse Strain ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Considerable Improvement ◽  
Es Cell ◽  
Partial Explanation ◽  
Developmental Potential ◽  
Skeletal Abnormalities ◽  
Embryonic Tissues

Diploid androgenetic eggs contain two sperm-derived genomes, and only rarely develop to the early somite stage. Also, previous studies have indicated that androgenetic eggs cannot be rescued in aggregation chimeras beyond embryonic stages. Paradoxically, in blastocyst injection chimeras made with androgenetic embryonic stem (ES) cells of the 129/Sv strain, we previously obtained considerable improvement in developmental potential. Although considerable death occurred in utero, overtly normal chimeric fetuses and occasional postnatal chimeras that developed skeletal abnormalities were observed. Consequently, we have re-evaluated the developmental potential of androgenetic aggregation chimeras utilizing androgenetic eggs of the 129/Sv strain, and of the BALB/c and CD-1 strains for comparison. Regardless of strain, androgenetic aggregation chimeras were generally more inviable than previously observed with androgenetic ES cell chimeras, and often the embryoproper was abnormal even when an androgenetic contribution was detected only in the extra-embryonic membranes. This is at least a partial explanation of the greater viability of androgenetic ES cell chimeras, as ES cells do not colonize significantly certain extra-embryonic tissues. Nevertheless, in the 129/Sv strain, occasional development of chimeras to term was obtained, and one chimera that survived postnatally developed identical skeletal abnormalities to those observed previously in androgenetic ES cell chimeras. This result demonstrates that at least one example of paternal imprinting is faithfully conserved in androgenetic ES cells. Also, the postnatal chimerism shows that androgenetic eggs can give rise to terminally differentiated cell types, and are therefore pluripotent. In contrast, only possibly one BALB/c and no CD-1 androgenetic aggregation chimeras developed to term. Therefore, the developmental potential of androgenetic aggregation chimeras is to some extent dependent on mouse strain.

182. NOVEL SIGNALLING IN MOUSE EMBRYONIC STEM CELLS GENERATES PRIMITIVE ECTODERM-LIKE CELLS

2009 ◽  
Vol 21 (9) ◽  
pp. 100
Author(s):  
M. B. Morris ◽  
N. Hamra ◽  
A. C. Lonic ◽  
F. Felquer
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Leukaemia Inhibitory Factor ◽  
Signalling Pathways ◽  
Specific Cell ◽  
Self Renewal ◽  
Mouse Es Cells ◽  
Homogeneous Production ◽  
Extracellular Molecules

The phenotypic status of embryonic stem (ES) cells is controlled in part by signalling pathways which translate inputs mediated by extracellular molecules. An important extracellular protagonist in mouse ES cells is LIF (leukaemia inhibitory factor) which interacts with the gp130–LIFR receptor complex to activate a number of downstream signalling pathways, including the STAT3, MEK/ERK and PI3K/Akt. These pathways, together with others, interact in complex and sometimes competing ways to generate the well-known characteristics of mouse ES cells of self-renewal, high rates of proliferation, and pluripotence. The addition of a second molecule, L-proline, to the extracellular environment alters the pluripotent status of mouse ES cells, converting them to a second pluripotent population equivalent to the primitive ectoderm of the pre-gastrulating embryo. This conversion, from ES cells to primitive ectoderm-like cells, primes the latter for directed differentiation to specific cell types (1). Here we show, using inhibitor studies and kinome array analysis, that this small molecule appears to work by (i) changing the balance in activity of signalling pathways already stimulated by LIF and (ii) activating additional signalling pathways. Specifically, L-proline rapidly further activates the LIF-stimulated MEK/ERK pathway, tipping the balance in favour of primitive-ectoderm formation and away from ES-cell self-renewal sustained by LIF-mediated activation of the STAT3 pathway. In addition, L-proline rapidly stimulates other pathways including p38, mTOR and PI3K/Akt each of which contributes, to a greater or lesser extent, to the conversion to primitive ectoderm-like cells. These results indicate that (i) L-proline acts in novel ways to stimulate embryo-like developmental progression in ES cells and (ii) through the addition of small, nontoxic activators and inhibitors of signalling pathways, the differentiation of pluripotent ES cells might be controlled sufficiently well for the homogeneous production of specific cell types suitable for use in animal models of human disease.

scholarly journals T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal

2012 ◽  
Vol 302 (3) ◽  
pp. C494-C504 ◽  
Author(s):  
José A. Rodríguez-Gómez ◽  
Konstantín L. Levitsky ◽  
José López-Barneo
Keyword(s):  
Cell Cycle ◽  
Alkaline Phosphatase ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Mrna Levels ◽  
Es Cell ◽  
External Application ◽  
Self Renewal ◽  
Mouse Es Cells

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na+ currents as well as transient Ca2+ currents abolished by the external application of Ni2+. Biophysical and pharmacological data indicated that the Ca2+ current is predominantly mediated by T-type (Cav3.2) channels. The number of cells expressing T-type channels and Cav3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca2+ currents with Ni2+ induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Cav3.2) Ca2+ channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Cav3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca2+ entry mediated by Cav3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.

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