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Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines.BioTechniques Nov 2000Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to...
Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.
Topics: Animals; Blastocyst; Cell Aggregation; Cell Culture Techniques; Cells, Cultured; Chimera; Clone Cells; Female; Germ-Line Mutation; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Transgenic; Microinjections; Morula; Stem Cells
PubMed: 11084865
DOI: 10.2144/00295st04 -
PloS One 2014When primary cultures of normal cells are cloned, three types of colony grow, called holoclones, meroclones and paraclones. These colonies are believed to be derived...
When primary cultures of normal cells are cloned, three types of colony grow, called holoclones, meroclones and paraclones. These colonies are believed to be derived from stem cells, transit-amplifying cells and differentiated cells respectively. More recently, this approach has been extended to cancer cell lines. However, we observed that meroclones from the prostate cancer cell line DU145 produce holoclones, a paradoxical observation as meroclones are thought to be derived from transit-amplifying cells. The purpose of this study was to confirm this observation and determine if both holoclones and meroclones from cancer cell lines contain stem cells. We demonstrated that both holoclones and meroclones can be serially passaged indefinitely, are highly proliferative, can self-renew to form spheres, are serially tumorigenic and express stem cell markers. This study demonstrates that the major difference between holoclones and meroclones derived from a cancer cell line is the proportion of stem cells within each colony, not the presence or absence of stem cells. These findings may reflect the properties of cancer as opposed to normal cells, perhaps indicating that the hierarchy of stem cells is more extensive in cancer.
Topics: Analysis of Variance; Biomarkers; Cell Differentiation; Cell Line, Tumor; Clone Cells; Colony-Forming Units Assay; Fluorescence; Humans; Immunohistochemistry; Male; Prostatic Neoplasms; Stem Cells
PubMed: 24587067
DOI: 10.1371/journal.pone.0089834 -
Developmental Immunology 1991Five stromal-cell-dependent lymphocyte clones are described that correspond to late pre-B or early B-cell stages of differentiation. They are useful for determining the...
Five stromal-cell-dependent lymphocyte clones are described that correspond to late pre-B or early B-cell stages of differentiation. They are useful for determining the molecular requirements for pre-B replication, for studying the stromal cells that supply those factors, and for delineating the final sequence of differentiation events as newly formed lymphocytes prepare to exit the bone marrow. The efficiency of lymphocyte growth at limiting dilution varied substantially on different stromal-cell clones and may reflect functional heterogeneity of stromal cells. Most lymphocyte clones were similar to uncloned lymphocytes from Whitlock-Witte cultures in that they responded only transiently to interleukin-7 (IL-7) and then died, unless maintained on a stromal-cell clone. One unusual lymphocyte clone (2E8) was propagated for more than 1 year in IL-7 alone and was selectively responsive to that cytokine. Most of the lymphocyte clones were not tumorigenic in immunodeficient mice. However, one pre-B clone (1A9) grew autonomously in culture when held at high density, responded to conditioned medium from a number of cell lines, and was tumorigenic. Tumors derived from this clone were infiltrated by stromal cells and lymphocytes taken from the tumors' retained characteristics of the original clone. Ly-6 antigens were inducible on 2E8 and 1A9 cells, but the lymphocytes were otherwise arrested in differentiation. The 2E8 cells had rearranged and expressed kappa light-chain genes but displayed them on the surface along with surrogate light chains and mu heavy chains. Thus, expression of authentic light chain need not coincide with termination of surrogate light-chain utilization in newly formed B cells. Several glycoproteins have recently been demonstrated to be associated with surface immunoglobulin (Ig) on mature B-lineage cells and plasma-cell tumors. We now show that one member of this family (approximately 33 kD) was associated with the mu+surrogate light-chain complex on the 1A9 pre-B-cell clone. When compared to mature B lymphomas, fewer bands coprecipitated with the surface-labeled Ig isolated from pre-B- and early B-cell lines, suggesting that components of the antigen receptor are sequentially acquired during development. The normal replication and differentiation of pre-B cells is probably regulated by complex interactions with multiple cytokines and matrix components of the marrow microenvironment. Cloned lymphocyte lines that are dependent on stromal cells should continue to be important tools for molecular definition of those interactions.
Topics: Animals; Antigens, Differentiation, B-Lymphocyte; B-Lymphocyte Subsets; Cell Differentiation; Cell Transformation, Neoplastic; Clone Cells; Cytokines; Gene Expression; Gene Rearrangement, B-Lymphocyte; Immunocompromised Host; Immunoglobulin Light Chains; Immunoglobulin Light Chains, Surrogate; Immunoglobulin mu-Chains; Interleukin-7; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Neoplasms, Experimental; Proto-Oncogenes
PubMed: 1821694
DOI: 10.1155/1991/79721 -
BMJ (Clinical Research Ed.) Jan 1995
Topics: Clone Cells; Dosage Compensation, Genetic; Female; Histiocytosis, Langerhans-Cell; Humans; Langerhans Cells; Mutation
PubMed: 7833724
DOI: 10.1136/bmj.310.6972.74 -
Biomolecules Dec 2021The BALB/c cell transformation assay (BALB-CTA) considers inter- and intra-tumor heterogeneities and affords the possibility of a direct comparison between untransformed...
The BALB/c cell transformation assay (BALB-CTA) considers inter- and intra-tumor heterogeneities and affords the possibility of a direct comparison between untransformed and malignant cells. In the present study, we established monoclonal cell lines that originate from the BALB-CTA and mimic heterogeneous tumor cell populations, in order to investigate phenotype-specific effects of the anti-diabetic drug metformin and the short-chain fatty acid butyrate. Growth inhibitory effects were measured with a ViCell XR cell counter. The BALB/c tumor therapy model (BALB-TTM) was performed, and the extracellular glucose level was measured in the medium supernatant. Using a Seahorse Analyzer, the metabolic phenotypes of four selected clones were characterized, and effects on energy metabolism were investigated. Anti-carcinogenic effects and reduced glucose uptake after butyrate application were observed in the BALB-TTM. Metabolic characterization of the cell clones revealed three different phenotypes. Surprisingly, treatment with metformin or butyrate induced opposite metabolic shifts with similar patterns in all cell clones tested. In conclusion, the BALB-TTM is a relevant model for mechanistic cancer research, and the generation of monoclonal cell lines offers a novel possibility to investigate specific drug effects in a heterogeneous tumor cell population. The results indicate that induced alterations in energy metabolism seem to be independent of the original metabolic phenotype.
Topics: Animals; Butyrates; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Clone Cells; Culture Media; Energy Metabolism; Glucose; Humans; Metformin; Mice; Models, Biological; Phenotype
PubMed: 34944475
DOI: 10.3390/biom11121831 -
The Journal of Reproduction and... Feb 2010It has now been 13 years since the first cloned mammal Dolly the sheep was generated from somatic cells using nuclear transfer (SCNT). Since then, this technique has... (Review)
Review
It has now been 13 years since the first cloned mammal Dolly the sheep was generated from somatic cells using nuclear transfer (SCNT). Since then, this technique has been considered an important tool not only for animal reproduction but also for regenerative medicine. However, the success rate is still very low and the mechanisms involved in genomic reprogramming are not yet clear. Moreover, the NT technique requires donated fresh oocyte, which raises ethical problems for production of human cloned embryo. For this reason, the use of induced pluripotent stem cells for genomic reprogramming and for regenerative medicine is currently a hot topic in this field. However, we believe that the NT approach remains the only valid way for the study of reproduction and basic biology. For example, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, and it can generate offspring from a single cell or even a frozen dead body. Thanks to much hard work by many groups, cloning success rates are increasing slightly year by year, and NT cloning is now becoming a more applicable method. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.
Topics: Animals; Cellular Reprogramming; Clone Cells; Cloning, Organism; Embryonic Stem Cells; Epigenesis, Genetic; Extinction, Biological; Female; Mice; Nuclear Transfer Techniques; Oocytes; Pluripotent Stem Cells
PubMed: 20203432
DOI: 10.1262/jrd.09-221a -
Blood Apr 2020In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating... (Clinical Trial)
Clinical Trial
In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.
Topics: Cell Differentiation; Cell Tracking; Clone Cells; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Hemoglobinopathies; Humans; Wiskott-Aldrich Syndrome
PubMed: 32040546
DOI: 10.1182/blood.2019002350 -
Science Immunology Nov 2018Natural killer (NK) cells recognize and eliminate infected and malignant cells. Their life histories are poorly understood, particularly in humans, due to lack of...
Natural killer (NK) cells recognize and eliminate infected and malignant cells. Their life histories are poorly understood, particularly in humans, due to lack of informative models and endogenous clonal markers. Here, we apply transplantation of barcoded rhesus macaque hematopoietic cells to interrogate the landscape of NK cell production, expansion, and life histories at a clonal level long term and after proliferative challenge. We identify oligoclonal populations of rhesus CD56CD16 NK cells that are characterized by marked expansions and contractions over time yet remained long-term clonally uncoupled from other hematopoietic lineages, including CD56CD16 NK cells. Individual or groups of CD56CD16 expanded clones segregated with surface expression of specific killer immunoglobulin-like receptors. These clonally distinct NK cell subpopulation patterns persisted for more than 4 years, including after transient in vivo anti-CD16-mediated depletion and subsequent regeneration. Profound and sustained interleukin-15-mediated depletion was required to generate new oligoclonal CD56CD16 NK cells. Together, our results indicate that linear NK cell production from multipotent hematopoietic progenitors or less mature CD56CD16 cells is negligible during homeostasis and moderate proliferative stress. In such settings, peripheral compartmentalized self-renewal can maintain the composition of distinct, differentiated NK cell subpopulations.
Topics: Animals; Clone Cells; Killer Cells, Natural; Macaca mulatta
PubMed: 30389798
DOI: 10.1126/sciimmunol.aat9781 -
Seminars in Hematology Feb 2024Immune surveillance mechanisms play a crucial role in maintaining lifelong immune homeostasis in response to pathologic stimuli and aberrant cell states. However, their...
Immune surveillance mechanisms play a crucial role in maintaining lifelong immune homeostasis in response to pathologic stimuli and aberrant cell states. However, their persistence, especially in the context of chronic antigenic exposure, can create a fertile ground for immune evasion. These escaping cell phenotypes, harboring a variety of genomic and transcriptomic aberrances, chiefly in human leukocyte antigen (HLA) and antigen presentation machinery genes, may survive and proliferate, featuring a scenario of clonal cell expansion with immune failure characteristics. While well characterized in solid and, to some extent, hematological malignancies, little is known about their occurrence and significance in other disease contexts. Historical literature highlights the role for escaping HLA-mediated recognition as a strategy adopted by virus to evade from the immune system, hinting at the potential for immune aberrant cell expansion in the context of chronic infections. Additionally, unmasked in idiopathic aplastic anemia as a mechanism able to rescue failing hematopoiesis, HLA clonal escape may operate in autoimmune disorders, particularly in tissues targeted by aberrant immune responses. Furthermore, senescent cell status emerging as immunogenic phenotypes stimulating T cell responses, may act as a bottleneck for the selection of such immune escaping clones, blurring the boundaries between neoplastic transformation, aging and inflammation. Here we provide a fresh overview and perspective on this immune-driven clonal cell expansion, linking pathophysiological features of neoplastic, autoimmune, infectious and senescence processes exposed to immune surveillance.
Topics: Humans; Autoimmunity; Anemia, Aplastic; Neoplasms; Autoimmune Diseases; HLA Antigens; Clone Cells
PubMed: 38341340
DOI: 10.1053/j.seminhematol.2024.01.002 -
Folia Biologica 2021Over the past decades, the in vitro use of pluripotent cell lines gained a crucial role in toxicology, preclinical drug testing and developmental biology. NTERA2 clone...
Over the past decades, the in vitro use of pluripotent cell lines gained a crucial role in toxicology, preclinical drug testing and developmental biology. NTERA2 clone D1 cells were identified as pluripotent cells with high potential for neural differentiation. Although they are commonly used cellular sources in neuropharmacology and neurodevelopmental studies, their endodermal and mesodermal differentiation potential awaits further characterization. Here, we devised improved protocols for hepatogenic and osteogenic differentiation of NTERA2 clone D1 cells. Our in vitro differentiation assays showed significant up-regulation of multiple hepatogenic markers. We also observed robust mineralization and osteogenic marker expression of NTERA2 clone D1 cells upon in vitro osteogenic induction. These results suggest that NTERA2 clone D1 cells may be utilized as an in vitro model system to study various aspects of liver biology and osteogenesis. In addition, tri-lineage differentiation of NTERA2 clone D1 cells may serve as a simple experimental control system when validating pluripotency of other cell types.
Topics: Cell Differentiation; Cell Line; Clone Cells; Liver; Osteogenesis
PubMed: 35439850
DOI: No ID Found