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 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 Oct-3/4 Maintains the Proliferative Embryonic Stem Cell State via Specific Binding to a Variant Octamer Sequence in the Regulatory Region of the UTF1 Locus

2005 ◽  
Vol 25 (12) ◽  
pp. 5084-5094 ◽  
Author(s):  
Masazumi Nishimoto ◽  
Satoru Miyagi ◽  
Toshiyuki Yamagishi ◽  
Takehisa Sakaguchi ◽  
Hitoshi Niwa ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Stem Cell ◽  
Molecular Mechanisms ◽  
Specific Binding ◽  
Es Cells ◽  
Regulatory Region ◽  
Embryonic Stem ◽  
Cell State ◽  
Teratoma Formation ◽  
Stem Cell State

ABSTRACT The POU transcription factor Oct-3/4 has been shown to be critical for maintaining embryonic stem (ES) cell character. However, the molecular mechanisms underlying its function remain elusive. We have previously shown that among the POU transcription factor family of proteins, Oct-3/4 alone is able to bind to the regulatory region of the UTF1 gene bearing a variant octamer sequence together with Sox-2. Here, we demonstrate using Oct-3/4-Oct-6 chimeras that there is a precise correlation between the ability of proteins to form a complex on the UTF1 enhancer with Sox-2 and the ability to maintain the stem cell state in ES cells. Different chimeric proteins show differential abilities to form a Sox-2-containing complex on the UTF1 regulatory region, with a decrease in efficiency of the complex formation accompanied by a decrease in the level of UTF1 expression and the rate of cell proliferation. Overexpression of UTF1 in these slow-growing cells was able to restore their proliferation rate to wild-type levels. Moreover, UTF1 was also observed to have an effect on teratoma formation. These results suggest a molecular pathway by which Oct-3/4 induces rapid proliferation and tumorigenic properties of ES cells through activation of the UTF1 gene.

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.

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.

In vitro differentiation of reprogrammed murine somatic cells into hepatic precursor cells

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Tobias Cantz ◽  
Martina Bleidißel ◽  
Martin Stehling ◽  
Hans R. Schöler
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Es Cells ◽  
Marrow Cell ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Precursor Cells ◽  
Differentiation Potential ◽  
Teratoma Formation ◽  
Transplantation Experiments

Abstract Recently, a new approach to reprogram somatic cells into pluripotent stem cells was shown by fusion of somatic cells with embryonic stem (ES) cells, which results in a tetraploid karyotype. Normal hepatocytes are often polyploid, so we decided to investigate the differentiation potential of fusion hybrids into hepatic cells. We chose toxic milk mice (a model of Wilson's disease) and performed initial transplantation experiments using this potential cell therapy approach. Mononuclear bone marrow cells from Rosa26 mice were fused with OG2 (Oct4-GFP transgenic) ES cells. Unfused ES cells were eliminated by selection with G418 for OG2-Rosa26 hybrids and fusion-derived colonies could be subcloned. Using an endodermal differentiation protocol, hepatic precursor cells could be generated. After FACS depletion of contaminating Oct4-GFP-positive cells, the hepatic precursor cells were transplanted into immunosuppressed toxic milk mice by intrasplenic injection. However, five out of eight mice showed teratoma formation within 3–6 weeks after transplantation in the spleen and liver. In conclusion, a hepatic precursor cell type was achieved from mononuclear bone marrow cell-ES cell hybrids and preliminary transplantation experiments confirmed engraftment, but also showed teratoma formation, which needs to be excluded by using more stringent purification strategies.

scholarly journals MEKK1 suppresses oxidative stress-induced apoptosis of embryonic stem cell-derived cardiac myocytes

1999 ◽  
Vol 96 (26) ◽  
pp. 15127-15132 ◽  
Author(s):  
T. Minamino ◽  
T. Yujiri ◽  
P. J. Papst ◽  
E. D. Chan ◽  
G. L. Johnson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cardiac Myocytes ◽  
Embryonic Stem ◽  
Induced Apoptosis

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).

431. Importin proteins and their role in maintenance of the stem cell state

2008 ◽  
Vol 20 (9) ◽  
pp. 111
Author(s):  
J. C. Young ◽  
Y. Miyamoto ◽  
A. Major ◽  
V. L. Dias ◽  
D. A. Jans ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Embryonic Stem ◽  
Distribution Patterns ◽  
Embryoid Bodies ◽  
Dominant Negative ◽  
Subcellular Localisation ◽  
Mrna Levels ◽  
Pluripotency Marker

The importin (IMP) family of proteins mediates transport into the nucleus for many proteins larger than 40 kD. Through differential cargo recognition, IMPs regulate cellular events by controlling nuclear access of transcription factors and chromatin remodelling agents. During spermatogenesis, many IMPs change expression and localisation in a manner concordant with specific stages of spermatogenic development. To assess the potential role of IMPs in the transition between the stem cell and subsequent differentiation, we undertook analysis of the expression and subcellular localisation of several key murine IMPs in both pluripotent embryonic stem cells (mESCs) and embryoid bodies (EBs). All of the IMPs analysed (IMPα2, 3, 4, IMPβ1 and IMP5) were detected in undifferentiated mESCs by immunofluorescence, and each exhibited distinctive nucleocytoplasmic distribution patterns. Subcellular localisation of most IMPs altered after 10 days of mESC differentiation as EBs. This was paralleled by changes in the mRNA levels of IMPα1–4, IMPα6, IMPβ1 and IMP5, concomitant with alterations in the expression level of the pluripotency marker, Oct3/4. Reducing IMP-dependent nuclear import through overexpression of specific dominant-negative IMP constructs led to alterations in import or production of Oct3/4 protein, depending on the specific IMP. These findings indicate that IMPs may play very specific but distinct roles in cell fate choice between maintenance of pluri/multipotency and commitment to differentiation in ESCs and potentially in spermatogenesis or other organs that contain stem cells.

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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