Sequences and factors required for the F9 embryonal carcinoma stem cell E1a-like activity

1991 ◽  
Vol 11 (11) ◽  
pp. 5534-5540
Author(s):  
E J Murray ◽  
D Stott ◽  
P W Rigby
Keyword(s):  
Stem Cells ◽  
Embryonal Carcinoma ◽  
Point Mutations ◽  
Cell Types ◽  
In Vitro Mutagenesis ◽  
Sequence Element ◽  
Cell Extracts ◽  
Transcription In Vitro ◽  
Efficient Expression

F9 embryonal carcinoma (EC) stem cells contain an E1a-like activity that is absent from differentiated derivatives. We have previously characterized proteins present in F9 EC cell extracts that bind to the E1a-dependent E2A promoter and have shown that two of them, TF68 and DRTF1, are required for efficient transcription in vitro (N. B. La Thangue, B. Thimmapaya, and P. W. J. Rigby, Nucleic Acids Res. 18:2929-2938, 1990). We now show that the E1a-like activity is detectable in transient transfection assays. Deletion mutations show that a distal sequence element, which includes the ATF/CREB consensus, is required for expression in both cell types, although it does not mediate the down-regulation of promoter activity that accompanies differentiation. A series of point mutations generated by in vitro mutagenesis confirm this and show that sequences around -60 are necessary for efficient expression in stem cells but not in differentiated derivatives. These sequences bind DRTF1, the activity of which is strongly down-regulated during differentiation. Surprisingly, mutations in a previously uncharacterized region of the promoter restore activity to a promoter carrying the -60 mutation and lead to the formation of a new DNA-protein complex.

scholarly journals Sequences and factors required for the F9 embryonal carcinoma stem cell E1a-like activity.

1991 ◽  
Vol 11 (11) ◽  
pp. 5534-5540 ◽  
Author(s):  
E J Murray ◽  
D Stott ◽  
P W Rigby
Keyword(s):  
Stem Cells ◽  
Embryonal Carcinoma ◽  
Point Mutations ◽  
Cell Types ◽  
In Vitro Mutagenesis ◽  
Sequence Element ◽  
Cell Extracts ◽  
Transcription In Vitro ◽  
Efficient Expression

F9 embryonal carcinoma (EC) stem cells contain an E1a-like activity that is absent from differentiated derivatives. We have previously characterized proteins present in F9 EC cell extracts that bind to the E1a-dependent E2A promoter and have shown that two of them, TF68 and DRTF1, are required for efficient transcription in vitro (N. B. La Thangue, B. Thimmapaya, and P. W. J. Rigby, Nucleic Acids Res. 18:2929-2938, 1990). We now show that the E1a-like activity is detectable in transient transfection assays. Deletion mutations show that a distal sequence element, which includes the ATF/CREB consensus, is required for expression in both cell types, although it does not mediate the down-regulation of promoter activity that accompanies differentiation. A series of point mutations generated by in vitro mutagenesis confirm this and show that sequences around -60 are necessary for efficient expression in stem cells but not in differentiated derivatives. These sequences bind DRTF1, the activity of which is strongly down-regulated during differentiation. Surprisingly, mutations in a previously uncharacterized region of the promoter restore activity to a promoter carrying the -60 mutation and lead to the formation of a new DNA-protein complex.

scholarly journals Multicomponent differentiation-regulated transcription factors in F9 embryonal carcinoma stem cells.

1991 ◽  
Vol 11 (3) ◽  
pp. 1686-1695 ◽  
Author(s):  
M K Shivji ◽  
N B La Thangue
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Dna Binding ◽  
Embryonal Carcinoma ◽  
Regulatory Sequence ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Dna Sequence Specificity ◽  
Cell Extracts

Murine F9 embryonal carcinoma (F9 EC) stem cells have an E1a-like transcription activity that is down-regulated as these cells differentiate to parietal endoderm. For the adenovirus E2A promoter, this activity requires at least two sequence-specific transcription factors, one that binds the cyclic AMP-responsive element (CRE) and the other, DRTF1, the DNA-binding activity of which is down-regulated as F9 EC cells differentiate. Here we report the characterization of several binding activities in F9 EC cell extracts, referred to as DRTF 1a, 1b and 1c, that recognize the DRTF1 cis-regulatory sequence (-70 to -50 region). These activities can be chromatographically separated but are not distinguishable by DNA sequence specificity. Activity 1a is a detergent-sensitive complex in which DNA binding is regulated by phosphorylation. In contrast, activities 1b and 1c are unaffected by these treatments but exist as multicomponent protein complexes even before DNA binding. Two sets of DNA-binding polypeptides, p50DR and p30DR, affinity purified from F9 EC cell extracts produce complexes 1b and 1c. Both polypeptides appear to be present in the same DNA-bound protein complex and both directly contact DNA. These affinity-purified polypeptides activate transcription in vitro in a binding-site-dependent manner. These data indicate the in F9 EC stem cells, multicomponent differentiation-regulated transcription factors contribute to the cellular E1a-like activity.

Neural Development by Transplanted Human Embryonal Carcinoma Stem Cells Expressing Green Fluorescent Protein

2005 ◽  
Vol 14 (6) ◽  
pp. 339-351 ◽  
Author(s):  
R. Stewart ◽  
M. Lako ◽  
G. M. Horrocks ◽  
S. A. Przyborski
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Neural Development ◽  
Molecular Mechanisms ◽  
Embryonal Carcinoma ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Green Fluorescent

For many years, researchers have investigated the fate and potential of neuroectodermal cells during the development of the central nervous system. Although several key factors that regulate neural differentiation have been identified, much remains unknown about the molecular mechanisms that control the fate and specification of neural subtypes, especially in humans. Human embryonal carcinoma (EC) stem cells are valuable research tools for the study of neural development; however, existing in vitro experiments are limited to inducing the differentiation of EC cells into only a handful of cell types. In this study, we developed and characterized a novel EC cell line (termed TERA2.cl.SP12-GFP) that carries the reporter molecule, green fluorescent protein (GFP). We demonstrate that TERA2.cl.SP12-GFP stem cells and their differentiated neural derivatives constitutively express GFP in cells grown both in vitro and in vivo. Cellular differentiation does not appear to be affected by insertion of the transgene. We propose that TERA2.cl.SP12-GFP cells provide a valuable research tool to track the fate of cells subsequent to transplantation into alternative environments and that this approach may be particularly useful to investigate the differentiation of human neural tissues in response to local environmental signals.

scholarly journals Multicomponent differentiation-regulated transcription factors in F9 embryonal carcinoma stem cells

1991 ◽  
Vol 11 (3) ◽  
pp. 1686-1695
Author(s):  
M K Shivji ◽  
N B La Thangue
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Dna Binding ◽  
Embryonal Carcinoma ◽  
Regulatory Sequence ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Dna Sequence Specificity ◽  
Cell Extracts

Murine F9 embryonal carcinoma (F9 EC) stem cells have an E1a-like transcription activity that is down-regulated as these cells differentiate to parietal endoderm. For the adenovirus E2A promoter, this activity requires at least two sequence-specific transcription factors, one that binds the cyclic AMP-responsive element (CRE) and the other, DRTF1, the DNA-binding activity of which is down-regulated as F9 EC cells differentiate. Here we report the characterization of several binding activities in F9 EC cell extracts, referred to as DRTF 1a, 1b and 1c, that recognize the DRTF1 cis-regulatory sequence (-70 to -50 region). These activities can be chromatographically separated but are not distinguishable by DNA sequence specificity. Activity 1a is a detergent-sensitive complex in which DNA binding is regulated by phosphorylation. In contrast, activities 1b and 1c are unaffected by these treatments but exist as multicomponent protein complexes even before DNA binding. Two sets of DNA-binding polypeptides, p50DR and p30DR, affinity purified from F9 EC cell extracts produce complexes 1b and 1c. Both polypeptides appear to be present in the same DNA-bound protein complex and both directly contact DNA. These affinity-purified polypeptides activate transcription in vitro in a binding-site-dependent manner. These data indicate the in F9 EC stem cells, multicomponent differentiation-regulated transcription factors contribute to the cellular E1a-like activity.

scholarly journals Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 876
Author(s):  
Raquel Bernad ◽  
Cian J. Lynch ◽  
Rocio G. Urdinguio ◽  
Camille Stephan-Otto Attolini ◽  
Mario F. Fraga ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Normal Karyotype ◽  
Cell Types ◽  
Dna Demethylation ◽  
Starting State ◽  
Teratoma Formation

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

scholarly journals Utilising Induced Pluripotent Stem Cells in Neurodegenerative Disease Research: Focus on Glia

2021 ◽  
Vol 22 (9) ◽  
pp. 4334
Author(s):  
Katrina Albert ◽  
Jonna Niskanen ◽  
Sara Kälvälä ◽  
Šárka Lehtonen
Keyword(s):  
Stem Cells ◽  
Neurodegenerative Diseases ◽  
Neurodegenerative Disease ◽  
Induced Pluripotent Stem Cells ◽  
Glial Cells ◽  
Pluripotent Stem Cells ◽  
Cell Types ◽  
Human Ipscs ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism’s somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer’s disease and Parkinson’s disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

scholarly journals Adipose-derived stromal/stem cells improve epidermal homeostasis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mariko Moriyama ◽  
Shunya Sahara ◽  
Kaori Zaiki ◽  
Ayumi Ueno ◽  
Koichi Nakaoji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Culture System ◽  
Wound Repair ◽  
Cell Types ◽  
Skin Wound ◽  
Epidermal Keratinocytes ◽  
Stromal Stem Cells ◽  
Collagen Vitrigel

AbstractWound healing is regulated by complex interactions between the keratinocytes and other cell types including fibroblasts. Recently, adipose-derived mesenchymal stromal/stem cells (ASCs) have been reported to influence wound healing positively via paracrine involvement. However, their roles in keratinocytes are still obscure. Therefore, investigation of the precise effects of ASCs on keratinocytes in an in vitro culture system is required. Our recent data indicate that the epidermal equivalents became thicker on a collagen vitrigel membrane co-cultured with human ASCs (hASCs). Co-culturing the human primary epidermal keratinocytes (HPEK) with hASCs on a collagen vitrigel membrane enhanced their abilities for cell proliferation and adhesion to the membrane but suppressed their differentiation suggesting that hASCs could maintain the undifferentiated status of HPEK. Contrarily, the effects of co-culture using polyethylene terephthalate or polycarbonate membranes for HPEK were completely opposite. These differences may depend on the protein permeability and/or structure of the membrane. Taken together, our data demonstrate that hASCs could be used as a substitute for fibroblasts in skin wound repair, aesthetic medicine, or tissue engineering. It is also important to note that a co-culture system using the collagen vitrigel membrane allows better understanding of the interactions between the keratinocytes and ASCs.

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 B-1 lymphoid cells develop independently of Notch signaling during mouse embryonic development

2020 ◽  
Author(s):  
Nathalia Azevedo ◽  
Elisa Bertesago ◽  
Ismail Ismailoglu ◽  
Michael Kyba ◽  
Michihiro Kobayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Development ◽  
Blood Cell ◽  
Notch Signaling ◽  
Embryonic Stem ◽  
Cell Types ◽  
Lymphoid Cells ◽  
Lineage Specification ◽  
Hematopoietic Stem

AbstractThe in vitro generation from pluripotent stem cells (PSCs) of different blood cell types, in particular those that are not replenished by hematopoietic stem cells (HSCs) like fetal-derived tissue-resident macrophages and innate-like lymphocytes, is of a particular interest. In order to succeed in this endeavor, a thorough understanding of the pathway interplay promoting lineage specification for the different blood cell types is needed. Notch signaling is essential for the HSC generation and their derivatives, but its requirement for tissue-resident immune cells is unknown. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B-1 cells. Their Notch-independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling was needed for the emergence of B-2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dosage is critical for the different B-cell lineage specification and provides pivotal information for their in vitro generation from PSCs for therapeutic applications.

scholarly journals The role of the 5′-flanking sequence of a human tRNAGlu gene in modulation of its transcriptional activity in vitro

1990 ◽  
Vol 272 (3) ◽  
pp. 797-803 ◽  
Author(s):  
E S Gonos ◽  
J P Goddard
Keyword(s):  
Transcriptional Activity ◽  
Potential Model ◽  
Trna Gene ◽  
Deletion Mutants ◽  
Wild Type ◽  
Flanking Sequence ◽  
Cell Extracts ◽  
Transcription In Vitro

The role of a tRNA-like structure within the 5′-flanking sequence of a human tRNA(Glu) gene in the modulation of its transcription in vitro by HeLa cell extracts has been investigated using several deletion mutants of a recombinant of the gene which lacked part or all of the tRNA-like structure. The transcriptional efficiency of four mutants was the same as that of the wild-type recombinant, two mutants had decreased transcriptional efficiency, one was more efficient, and one, lacking part of the 5′ intragenic control region, was inactive. Correlation of the transcriptional efficiencies with the position and the size of the 5′-flanking sequence that was deleted indicated that the tRNA-like structure may be deleted without loss of transcriptional efficiency. Current models for the modulation of tRNA gene transcription by the 5′-flanking sequence are assessed in the light of the results obtained, and a potential model is presented.

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