scholarly journals TSH stimulates adipogenesis in mouse embryonic stem cells

2007 ◽  
Vol 196 (1) ◽  
pp. 159-169 ◽  
Author(s):  
Min Lu ◽  
Reigh-Yi Lin
Keyword(s):  
Adipocyte Differentiation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Binding Activity ◽  
Intracellular Camp ◽  
Directed Differentiation ◽  
Adipose Differentiation ◽  
Main Regulator ◽  
And Function

Although TSH is the main regulator of thyroid growth and function, TSH binding activity in fat has long been reported. Since the TSH receptor (TSHR) has been detected in both preadipocytes and adipocytes, we hypothesized that it may play a role in adipose differentiation. Here, we use an in vitro model of adipogenesis from mouse embryonic stem (ES) cells to define TSH function. Directed differentiation of ES cells into the adipose lineage can be achieved over a 3-week period. Although adipocyte differentiation is initiated early in the development of cultured ES cells, TSHR up-regulation is precisely correlated with terminal differentiation of those adipocytes. The adipocytes express TSHR on the cell surface and respond to TSH with increased intracellular cAMP production, suggesting the activation of the protein kinase A signaling pathway. To determine whether TSH impacts adipogenesis, we examined how adipocytes responded to TSH at various points during their differentiation from cultured ES cells. We found that TSH greatly increases adipogenesis when added in the presence of adipogenic factors. More importantly, our data suggest that TSH also stimulates adipogenesis in cultured ES cells even in the absence of adipogenic factors. This finding provides the first evidence of TSH being a pro-adipogenic factor that converts ES cells into adipocytes. It further highlights the potential of ES cells as a model system for use in the study of TSH's role in the regulation of physiologically relevant adipose tissue.

Expression and function of CD105 during the onset of hematopoiesis from Flk1+ precursors

Blood ◽  
2001 ◽  
Vol 98 (13) ◽  
pp. 3635-3642 ◽  
Author(s):  
Sarah K. Cho ◽  
Annie Bourdeau ◽  
Michelle Letarte ◽  
Juan Carlos Zúñiga-Pflücker
Keyword(s):  
Transforming Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transforming Growth Factor Β ◽  
Hematopoietic Development ◽  
Mesoderm Specification ◽  
Molecular Events ◽  
Regulated Expression ◽  
And Function

Abstract During ontogeny, the hematopoietic system is established from mesoderm-derived precursors; however, molecular events regulating the onset of hematopoiesis are not well characterized. Several members of the transforming growth factor β (TGF-β) superfamily have been implicated as playing a role during mesoderm specification and hematopoiesis. CD105 (endoglin) is an accessory receptor for members of the TGF-β superfamily. Here it is reported that during the differentiation of murine embryonic stem (ES) cells in vitro, hematopoietic commitment within Flk1+ mesodermal precursor populations is characterized by CD105 expression. In particular, CD105 is expressed during the progression from the Flk1+CD45− to Flk1−CD45+ stage. The developmentally regulated expression of CD105 suggests that it may play a role during early hematopoiesis from Flk1+ precursors. To determine whether CD105 plays a functional role during early hematopoietic development, the potential of CD105-deficient ES cells to differentiate into various hematopoietic lineages in vitro was assessed. In the absence of CD105, myelopoiesis and definitive erythropoiesis were severely impaired. In contrast, lymphopoiesis appeared to be only mildly affected. Thus, these findings suggest that the regulated expression of CD105 functions to support lineage-specific hematopoietic development from Flk1+ precursors.

scholarly journals Calreticulin reveals a critical Ca2+ checkpoint in cardiac myofibrillogenesis

2002 ◽  
Vol 158 (1) ◽  
pp. 103-113 ◽  
Author(s):  
Jian Li ◽  
Michel Pucéat ◽  
Carmen Perez-Terzic ◽  
Annabelle Mery ◽  
Kimitoshi Nakamura ◽  
...  
Keyword(s):  
Heart Development ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cardiac Cells ◽  
Embryoid Bodies ◽  
Cell Functions ◽  
Myosin Light Chain 2 ◽  
And Function

Calreticulin (crt) is an ubiquitously expressed and multifunctional Ca2+-binding protein that regulates diverse vital cell functions, including Ca2+ storage in the ER and protein folding. Calreticulin deficiency in mice is lethal in utero due to defects in heart development and function. Herein, we used crt−/− embryonic stem (ES) cells differentiated in vitro into cardiac cells to investigate the molecular mechanisms underlying heart failure of knockout embryos. After 8 d of differentiation, beating areas were prominent in ES-derived wild-type (wt) embryoid bodies (EBs), but not in ES-derived crt−/− EBs, despite normal expression levels of cardiac transcription factors. Crt−/− EBs exhibited a severe decrease in expression and a lack of phosphorylation of ventricular myosin light chain 2 (MLC2v), resulting in an impaired organization of myofibrils. Crt−/− phenotype could be recreated in wt cells by chelating extracellular or cytoplasmic Ca2+ with EGTA or BAPTA, or by inhibiting Ca2+/calmodulin-dependent kinases (CaMKs). An imposed ionomycin-triggered cystolic-free Ca2+ concentration ([Ca2+]c) elevation restored the expression, phosphorylation, and insertion of MLC2v into sarcomeric structures and in turn the myofibrillogenesis. The transcription factor myocyte enhancer factor C2 failed to accumulate into nuclei of crt−/− cardiac cells in the absence of ionomycin-triggered [Ca2+]c increase. We conclude that the absence of calreticulin interferes with myofibril formation. Most importantly, calreticulin deficiency revealed the importance of a Ca2+-dependent checkpoint critical for early events during cardiac myofibrillogenesis.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

scholarly journals The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

scholarly journals Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2870-2882 ◽  
Author(s):  
Unmesh Jadhav ◽  
J. Larry Jameson
Keyword(s):  
Target Genes ◽  
De Novo ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Steroidogenic Factor 1 ◽  
Lineage Differentiation ◽  
And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

scholarly journals Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.

1997 ◽  
Vol 17 (3) ◽  
pp. 1642-1651 ◽  
Author(s):  
M J Weiss ◽  
C Yu ◽  
S H Orkin
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Zinc Finger ◽  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Transactivation Domain ◽  
Stage Of Development ◽  
Erythroid Maturation

The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

Basic fibroblast growth factor positively regulates hematopoietic development

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1931-1941 ◽  
Author(s):  
P. Faloon ◽  
E. Arentson ◽  
A. Kazarov ◽  
C.X. Deng ◽  
C. Porcher ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Fibroblast Growth ◽  
Hematopoietic Lineage ◽  
Mesoderm Inducing Factors

Recently identified BLast Colony Forming Cells (BL-CFCs) from in vitro differentiated embryonic stem (ES) cells represent the common progenitor of hematopoietic and endothelial cells, the hemangioblast. Access to this initial cell population committed to the hematopoietic lineage provides a unique opportunity to characterize hematopoietic commitment events. Here, we show that BL-CFC expresses the receptor tyrosine kinase, Flk1, and thus we took advantage of the BL-CFC assay, as well as fluorescent activated cell sorter (FACS) analysis for Flk1(+) cells to determine quantitatively if mesoderm-inducing factors promote hematopoietic lineage development. Moreover, we have analyzed ES lines carrying targeted mutations for fibroblast growth factor receptor-1 (fgfr1), a receptor for basic fibroblast growth factor (bFGF), as well as scl, a transcription factor, for their potential to generate BL-CFCs and Flk1(+) cells, to further define events leading to hemangioblast development. Our data suggest that bFGF-mediated signaling is critical for the proliferation of the hemangioblast and that cells expressing both Flk1 and SCL may represent the hemangioblast.

scholarly journals Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells

2018 ◽  
Vol 19 (8) ◽  
pp. 2425 ◽  
Author(s):  
Orazio Angelo Arcidiacono ◽  
Jana Krejčí ◽  
Jana Suchánková ◽  
Eva Bártová
Keyword(s):  
Histone Deacetylases ◽  
Trichostatin A ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cardiomyocyte Differentiation ◽  
Histone H4 ◽  
Epigenetic Factors ◽  
Double Knockout ◽  
Physiological Processes

Cell differentiation into cardiomyocytes requires activation of differentiation-specific genes and epigenetic factors that contribute to these physiological processes. This study is focused on the in vitro differentiation of mouse embryonic stem cells (mESCs) induced into cardiomyocytes. The effects of clinically promising inhibitors of histone deacetylases (HDACi) on mESC cardiomyogenesis and on explanted embryonic hearts were also analyzed. HDAC1 depletion caused early beating of cardiomyocytes compared with those of the wild-type (wt) counterpart. Moreover, the adherence of embryonic bodies (EBs) was reduced in HDAC1 double knockout (dn) mESCs. The most important finding was differentiation-specific H4 deacetylation observed during cardiomyocyte differentiation of wt mESCs, while H4 deacetylation was weakened in HDAC1-depleted cells induced to the cardiac pathway. Analysis of the effect of HDACi showed that Trichostatin A (TSA) is a strong hyperacetylating agent, especially in wt mESCs, but only SAHA reduced the size of the beating areas in EBs that originated from HDAC1 dn mESCs. Additionally, explanted embryonic hearts (e15) responded to treatment with HDACi: all of the tested HDACi (TSA, SAHA, VPA) increased the levels of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines in embryonic hearts. This observation shows that explanted tissue can be maintained in a hyperacetylation state several hours after excision, which appears to be useful information from the view of transplantation strategy and the maintenance of gene upregulation via acetylation in tissue intended for transplantation.

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