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 Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell–derived neural transplants

2004 ◽  
Vol 167 (4) ◽  
pp. 723-734 ◽  
Author(s):  
Erhard Bieberich ◽  
Jeane Silva ◽  
Guanghu Wang ◽  
Kannan Krishnamurthy ◽  
Brian G. Condie
Keyword(s):  
Stem Cell ◽  
Neural Differentiation ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Human Stem Cells ◽  
Pluripotency Marker ◽  
Lineage Differentiation ◽  
Induced Apoptosis ◽  
Teratoma Formation

The formation of stem cell–derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body–derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of β-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

scholarly journals Identification and Characterisation of Putative Enhancer Elements in Mouse Embryonic Stem Cells

2021 ◽  
Vol 15 ◽  
pp. 117793222097462
Author(s):  
Anna Mantsoki ◽  
Karla Parussel ◽  
Anagha Joshi
Keyword(s):  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Data Sets ◽  
Simultaneous Presence ◽  
Enhancer Activity ◽  
Cellular Context ◽  
Distinct Sequence ◽  
A Cell ◽  
And Function

Enhancer elements control mammalian transcription largely in a cell-type-specific manner. The genome-wide identification of enhancer elements and their activity status in a cellular context is therefore fundamental to understanding cell identity and function. We determined enhancer activity in mouse embryonic stem (ES) cells using chromatin modifications and characterised their global properties. Specifically, we first grouped enhancers into 5 groups using multiple H3K4me1, H3K27ac, and H3K27me3 modification data sets. Active enhancers (simultaneous presence of H3K4me1 and H3K27ac) were enriched for binding of pluripotency factors and were found near pluripotency-related genes. Although both H3K4me1-only and active enhancers were enriched for super-enhancers and a TATA box like motif, active enhancers were preferentially bound by RNA polII (s2) and were enriched for bidirectional transcription, while H3K4me1-only enhancers were enriched for RNA polII (8WG16) suggesting they were likely poised. Bivalent enhancers (simultaneous presence of H3K4me1 and H3K27me3) were preferentially in the vicinity of bivalent genes. They were enriched for binding of components of polycomb complex as well as Tcf3 and Oct4. Moreover, a ‘CTTTCTC’ de-novo motif was enriched at bivalent enhancers, previously identified at bivalent promoters in ES cells. Taken together, 3 histone modifications successfully demarcated active, bivalent, and poised enhancers with distinct sequence and binding features.

Abstract 134: Rigid Microenvironments Promote Cardiac Differentiation Of Mouse And Human Embryonic Stem Cells

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Armin Arshi ◽  
Yasuhiro Nakashima ◽  
Haruko Nakano ◽  
Sarayoot Eaimkhong ◽  
Denis Evseenko ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cardiac Tissue ◽  
De Novo ◽  
Troponin T ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Cardiac Differentiation ◽  
Expression Ratio

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using an genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of the already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media

Development ◽  
2002 ◽  
Vol 129 (2) ◽  
pp. 539-549 ◽  
Author(s):  
Carrie A. Adelman ◽  
Subrata Chattopadhyay ◽  
James J. Bieker
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Dominant Negative ◽  
Erythroid Cell ◽  
Specific Gene ◽  
Smad Pathway ◽  
Cellular Expression ◽  
Novel Approach

Erythroid cell-specific gene regulation during terminal differentiation is controlled by transcriptional regulators, such as EKLF and GATA1, that themselves exhibit tissue-restricted expression patterns. Their early expression, already in evidence within multipotential hematopoietic cell lines, has made it difficult to determine what extracellular effectors and transduction mechanisms might be directing the onset of their own transcription during embryogenesis. To circumvent this problem, we have taken the novel approach of investigating whether the ability of embryonic stem (ES) cells to mimic early developmental patterns of cellular expression during embryoid body (EB) differentiation can address this issue. We first established conditions whereby EBs could form efficiently in the absence of serum. Surprisingly, in addition to mesoderm, these cells expressed hemangioblast and hematopoietic markers. However, they did not express the committed erythroid markers EKLF and GATA1, nor the terminally differentiated β-like globin markers. Using this system, we determined that EB differentiation in BMP4 was necessary and sufficient to recover EKLF and GATA1 expression and could be further stimulated by the inclusion of VEGF, SCF, erythropoietin and thyroid hormone. EBs were competent to respond to BMP4 only until day 4 of differentiation, which coincides with the normal onset of EKLF expression. The direct involvement of the BMP/Smad pathway in this induction process was further verified by showing that erythroid expression of a dominant negative BMP1B receptor or of the inhibitory Smad6 protein prevented induction of EKLF or GATA1 even in the presence of serum. Although Smad1, Smad5 and Smad8 are all expressed in the EBs, BMP4 induction of EKLF and GATA1 transcription is not immediate. These data implicate the BMP/Smad induction system as being a crucial pathway to direct the onset of EKLF and GATA1 expression during hematopoietic differentiation and demonstrate that EB differentiation can be manipulated to study induction of specific genes that are expressed early within a lineage.

Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2853-2859 ◽  
Author(s):  
A. Weng ◽  
T. Magnuson ◽  
U. Storb
Keyword(s):  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Mouse Development ◽  
Partial Methylation ◽  
Distal Chromosome ◽  
Before And After ◽  
Endogenous Genes ◽  
De Novo Methylation

A murine transgene, HRD, is methylated only when carried in certain inbred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain-specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryos revealed that strain-specific methylation is initiated prior to embryonic day (E) 6.5. Strain-specific transgene methylation is all-or-none in pattern and occurs exclusively in the primitive ectoderm lineage. A strain-independent pattern of partial methylation occurs in the primitive endoderm and trophectoderm lineages. To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and examined for transgene methylation before and after differentiation. Though the transgene had already acquired some methylation in undifferentiated ES cells, differentiation induced further, de novo methylation in a strain-dependent manner. Analysis of methylation in ES cultures suggests that the transgene and endogenous genes (such as immunoglobulin genes) are synchronously methylated during early development. These results are interpreted in the context of a model in which Ssm1-like modifier genes produce alterations in chromatin structure during and/or shortly after implantation, thereby marking target loci for de novo methylation with the rest of the genome during gastrulation.

scholarly journals Differences in Gene Expression between Wild Type and Hoxa1 Knockout Embryonic Stem Cells after Retinoic Acid Treatment or Leukemia Inhibitory Factor (LIF) Removal

2005 ◽  
Vol 280 (16) ◽  
pp. 16484-16498 ◽  
Author(s):  
Eduardo Martinez-Ceballos ◽  
Pierre Chambon ◽  
Lorraine J. Gudas
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Retinoic Acid ◽  
Leukemia Inhibitory Factor ◽  
Target Genes ◽  
Hox Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Inhibitory Factor ◽  
Hoxa Cluster

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. In embryos, alterations of the normal pattern of Hox gene expression result in homeotic transformations and malformations. Disruption of theHoxa1gene, the most 3′ member of the Hoxa cluster and a retinoic acid (RA) direct target gene, results in abnormal ossification of the skull, hindbrain, and inner ear deficiencies, and neonatal death. We have generated Hoxa1-/-embryonic stem (ES) cells (named Hoxa1-15) from Hoxa1-/-mutant blastocysts to study the Hoxa1 signaling pathway. We have characterized in detail these Hoxa1-/-ES cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1,Postn/Osf2, and the bone sialoprotein gene orBSP), genes that are expressed in the developing brain (e.g. Nnat,Wnt3a,BDNF,RhoB, andGbx2), and genes involved in various cellular processes (e.g. M-RAS,Sox17,Cdkn2b,LamA1,Col4a1,Foxa2,Foxq1,Klf5, andIgf2). Cell proliferation assays and Northern blot analyses of a number of ES cell markers (e.g. Rex1,Oct3/4,Fgf4, andBmp4) suggest that the Hoxa1 protein plays a role in the inhibition of cell proliferation by RA in ES cells. Additionally, Hoxa1-/-ES cells express high levels of various endodermal markers, includingGata4andDab2, and express much lessFgf5after leukemia inhibitory factor (LIF) withdrawal. Finally, we propose a model in which the Hoxa1 protein mediates repression of endodermal differentiation while promoting expression of ectodermal and mesodermal characteristics.

scholarly journals METTL3 Modulates Osteoclast Differentiation and Function by Controlling RNA Stability and Nuclear Export

2020 ◽  
Vol 21 (5) ◽  
pp. 1660 ◽  
Author(s):  
Di Li ◽  
Luhui Cai ◽  
Runsha Meng ◽  
Zhihui Feng ◽  
Qiong Xu
Keyword(s):  
Nuclear Export ◽  
Osteoclast Differentiation ◽  
Rna Stability ◽  
Cell Lineage ◽  
Specific Gene ◽  
Bone Homeostasis ◽  
Protein Levels ◽  
Lineage Differentiation ◽  
And Function ◽  
Skeletal Integrity

Osteoclast differentiation and function are crucial for maintaining bone homeostasis and preserving skeletal integrity. N6-methyladenosine (m6A) is an abundant mRNA modification that has recently been shown to be important in regulating cell lineage differentiation. Nevertheless, the effect of m6A on osteoclast differentiation remains unknown. In the present study, we observed that the m6A level and methyltransferase METTL3 expression increased during osteoclast differentiation. Mettl3 knockdown resulted in an increased size but a decreased bone-resorbing ability of osteoclasts. The expression of osteoclast-specific genes (Nfatc1, c-Fos, Ctsk, Acp5 and Dcstamp) was inhibited by Mettl3 depletion, while the expression of the cellular fusion-specific gene Atp6v0d2 was upregulated. Mechanistically, Mettl3 knockdown elevated the mRNA stability of Atp6v0d2 and the same result was obtained when the m6A-binding protein YTHDF2 was silenced. Moreover, the phosphorylation levels of key molecules in the MAPK, NF-κB and PI3K-AKT signaling pathways were reduced upon Mettl3 deficiency. Depletion of Mettl3 maintained the retention of Traf6 mRNA in the nucleus and reduced the protein levels of TRAF6. Taken together, our data suggest that METTL3 regulates osteoclast differentiation and function through different mechanisms involving Atp6v0d2 mRNA degradation mediated by YTHDF2 and Traf6 mRNA nuclear export. These findings elucidate the molecular basis of RNA epigenetic regulation in osteoclast development.

398 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO CARDIOMYOCYTES BY USING SLOW TURNING LATERAL VESSEL (STLV/BIOREACTOR)

2010 ◽  
Vol 22 (1) ◽  
pp. 355
Author(s):  
S. Rungarunlert ◽  
K. Tar ◽  
S. Muenthaisong ◽  
M. Techakumphu ◽  
M. Pirity ◽  
...  
Keyword(s):  
Large Scale ◽  
Embryoid Body ◽  
Troponin T ◽  
Es Cells ◽  
Statistical Significance ◽  
Embryonic Stem ◽  
Industrial Applications ◽  
Cardiac Differentiation ◽  
Seeding Density ◽  
Es Cell

Cardiomyocytes derived from embryonic stem (ES) cells are anticipated to be valuable for cardiovascular drug testing and disease therapies. The overall efficiency and quantity of cardiomyocytes obtained by differentiation of ES cells is still low. To enable a large-scale culture of ES-derived cells, we have tested a scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, bioreactor/STLV (slow turning lateral vessel, Synthecon, Inc., Houston, TX, USA) following inoculation with a single cell suspension of mouse ES cells. Technical parameters for optimal cell expansion and efficient ES cell differentiation were compared, such as ES cell seeding density (3 × 105 and 5 × 105 cells mL-1) into the bioreactor and day of transfer and plating of EB on gelatinated petri dishes (Day 2, Day 3, Day 4, and Day 5). The quantity and quality of EB production including the yield and size of EB, as well as viability and apoptosis of cells, were analyzed. Furthermore, after cultivation, well-developed contracting EB with functional cardiac muscle were obtained in which the percentage of EB beating/well and several specific cardiac genes [cardiac Troponin T (cTnT) and α-actinin] expression were also determined. Data are expressed as mean ± SEM of at least 3 independent experiments. Statistical analyses included one-way ANOVA and Student’s t-test Statistical significance was set at P < 0.05. The results showed that 5 × 105 ES cells mL-1 seeded into the STLV significantly improved the homogeneity of size of EB formed compared with 3 × 105 ES cells mL-1. The EB derived from Days 2 or 3 culturing in STLV had less necrotic cells than Days 4 and 5 groups. Furthermore, plating these EB on Days 2 and 3 resulted in significantly more EB beating/well than that of Days 4 and 5 groups. For cardiac differentiation, the group with 5 × 105 ES cells mL-1 seeded into STLV and transferred and plated on Day 3 expressed more cardiac markers than other groups. In conclusion, the optimized rotary suspension culture method can produce a highly uniform population of efficiently differentiating EB in large quantities in a manner that can be easily implemented by basic research laboratories. This method provides a technological platform for the controlled large-scale generation of ES cell-derived cells for clinical and industrial applications. This work was financed by The Thailand Commission on Higher Education (CHE-PhD-SW-2005-100), EUFP6 CLONET (MRTN-CT-2006-035468), NKFP_07_1-ES2HEART-HU (OM-00202-2007), and EUFP7 (PartnErS, PIAP-GA-2008-218205).

scholarly journals Hippo-YAP signaling controls lineage differentiation of mouse embryonic stem cells through modulating the formation of super-enhancers

2020 ◽  
Author(s):  
Xiang Sun ◽  
Zhijun Ren ◽  
Yixian Cun ◽  
Cai Zhao ◽  
Xianglin Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Signaling Pathway ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Hippo Signaling ◽  
Rna Seq ◽  
Lineage Differentiation ◽  
Super Enhancer

Abstract Hippo-YAP signaling pathway functions in early lineage differentiation of pluripotent stem cells, but the detailed mechanisms remain elusive. We found that knockout (KO) of Mst1 and Mst2, two key components of the Hippo signaling in mouse embryonic stem cells (ESCs), resulted in a disruption of differentiation into mesendoderm lineage. To further uncover the underlying regulatory mechanisms, we performed a series of ChIP-seq experiments with antibodies against YAP, ESC master transcription factors and some characterized histone modification markers as well as RNA-seq assays using wild type and Mst KO samples at ES and day 4 embryoid body stage respectively. We demonstrate that YAP is preferentially co-localized with super-enhancer (SE) markers such as Nanog, Sox2, Oct4 and H3K27ac in ESCs. The hyper-activation of nuclear YAP in Mst KO ESCs facilitates the binding of Nanog, Sox2 and Oct4 as well as H3K27ac modification at the loci where YAP binds. Moreover, Mst depletion results in novel SE formation and enhanced liquid-liquid phase-separated Med1 condensates on lineage associated genes, leading to the upregulation of these genes and the distortion of ESC differentiation. Our study reveals a novel mechanism on how Hippo-YAP signaling pathway dictates ESC lineage differentiation.

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