scholarly journals Valproic Acid Improves Porcine Parthenogenetic Embryo Development Through Transient Remodeling of Histone Modifiers

2015 ◽  
Vol 37 (4) ◽  
pp. 1463-1473 ◽  
Author(s):  
Yongye Huang ◽  
Lin Yuan ◽  
Tianye Li ◽  
Anfeng Wang ◽  
Zhanjun Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Histone Deacetylase Inhibitors ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Cell Stage ◽  
Alternative Source ◽  
Histone Modifiers ◽  
Parthenogenetic Embryos

Background/Aims: Parthenogenetic embryos are useful in many applications, such as being an alternative source of embryonic stem cells that would avoid ethical problems. Aberrance in epigenetic reprogramming is considered the major reason for the developmental failure of parthenogenetic embryos. Many histone deacetylase inhibitors have been shown to improve the reprogramming of stem cells and embryos. Here, the relationship between histone modification and parthenogenetic embryonic development was explored. Methods: Valproic acid (VPA) treatment was applied during the culture of parthenogenetic embryos. The abundance of histone modifiers was examined by immunofluorescence and quantified by Image-pro software. Results: The acH3K9 level in in vitro fertilized embryos was significantly higher than parthenogenetic embryos. VPA treatment improved both the blastocyst formation rate and the acH3K9 level in parthenogenetic embryos. The signal intensities of acH4K5 and H3K4me2 were also enhanced in VPA treated embryos. The H3K27me2 level was decreased in the VPA treated embryos at the 2-cell stage. However, the enhancement in the acH3K9, acH4K5 and H3K4me2 level, or the decrease in the H3K27me2 level disappeared shortly after VPA withdrawal. Conclusion: Optimizing histone modifications for a short time following activation was sufficient to enhance the in vitro development of parthenogenetic embryos.

47 DIFFERENTIAL DEVELOPMENTAL ABILITY OF EMBRYOS CLONED FROM TISSUE-SPECIFIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 142
Author(s):  
K. Inoue ◽  
N. Ogonuki ◽  
H. Miki ◽  
S. Noda ◽  
S. Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
High Rate ◽  
Full Potential ◽  
Cell Nuclei ◽  
Cell Stage

Although cloning animals by somatic cell nuclear transfer is generally an inefficient process, use of appropriate donor cell types may improve the cloning outcome significantly. Among the donor cells tested so far, mouse embryonic stem cells have given the best efficiency in terms of the development of reconstructed embryos into offspring. In this study, we examined whether 2 in vitro-produced pluripotent stem cells—neural stem cells (NSCs) and mesenchymal stem cells (MSCs)—could be better nuclear donors than other differentiated cells. Embryos were reconstructed by transfer of nuclei from NSCs or MSCs with full potential for differentiation in vitro. Most (76%) of the 2-cell NCS embryos developed to the 4-cell stage; 43% implanted and 1.6% developed to term after transfer to pseudopregnant recipients. These rates were very similar to those of embryos cloned from fibroblast cell nuclei. Interestingly, in the patterns of zygotic gene expression, NSC embryos were more similar to in vitro-fertilized embryos than fibroblast cloned embryos. By contrast, embryos reconstructed using MSC nuclei showed lower developmental ability and no implantation was obtained after embryo transfer. Chromosomal analysis of the donor MSCs revealed very high frequencies of monosomy and trisomy, which might have caused the very poor post-implantation development of embryos following nuclear transfer. Thus, in vitro-produced pluripotent cells can serve as donors of nuclei for cloning mice, but may be prone to chromosomal aberrations leading to a high rate of cloned embryo death.

scholarly journals Human mesenchymal stem cells derived from induced pluripotent stem cells down-regulate NK-cell cytolytic machinery

Blood ◽  
2011 ◽  
Vol 118 (12) ◽  
pp. 3254-3262 ◽  
Author(s):  
Massimo Giuliani ◽  
Noufissa Oudrhiri ◽  
Zaeem M. Noman ◽  
Amelia Vernochet ◽  
Salem Chouaib ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Nk Cell ◽  
Embryonic Stem ◽  
Attractive Alternative ◽  
Target Cells ◽  
Alternative Source ◽  
Activation Markers ◽  
Induced Pluripotent

Abstract A major issue in immunosuppressive biotherapy is the use of mesenchymal stem cells (MSCs) that harbor regulatory capacity. However, currently used bone marrow-derived MSCs (BM-MSCs) are short-lived and cannot assure long lasting immunoregulatory function both in vitro and in vivo. Consequently, we have generated MSCs from human induced pluripotent stem (IPS-MSCs) cells that share similar properties with embryonic stem cells (ES-MSCs). Herein, we compared the immunoregulatory properties of ES/IPS-MSCs with those of BM-MSCs and showed, for the first time, that IPS-derived MSCs display remarkable inhibition of NK-cell proliferation and cytolytic function in a similar way to ES-MSCs. Both MSCs disrupt NK-cell cytolytic machinery in the same fashion that BM-MSCs, by down-regulating the expression of different activation markers and ERK1/2 signaling, leading to an impairment to form immunologic synapses with target cells and, therefore, secretion of cytotoxic granules. In addition, they are more resistant than adult BM-MSCs to preactivated NK cells. IPS-MSCs could represent an attractive alternative source of immunoregulatory cells, and their capacity to impair NK-cell cytotoxicity constitutes a complex mechanism to prevent allograft rejection.

Hepatic differentiation of pluripotent stem cells

2009 ◽  
Vol 390 (10) ◽  
Author(s):  
Komal Loya ◽  
Reto Eggenschwiler ◽  
Kinarm Ko ◽  
Malte Sgodda ◽  
Francoise André ◽  
...  
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Pluripotent Stem Cells ◽  
Ethical Issues ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hepatic Differentiation ◽  
Alternative Source ◽  
Organ Specific

Abstract In regenerative medicine pluripotent stem cells are considered to be a valuable self-renewing source for therapeutic cell transplantations, given that a functional organ-specific phenotype can be acquired by in vitro differentiation protocols. Furthermore, derivatives of pluripotent stem cells that mimic fetal progenitor stages could serve as an important tool to analyze organ development with in vitro approaches. Because of ethical issues regarding the generation of human embryonic stem (ES) cells, other sources for pluripotent stem cells are intensively studied. Like in less developed vertebrates, pluripotent stem cells can be generated from the female germline even in mammals, via parthenogenetic activation of oocytes. Recently, testis-derived pluripotent stem cells were derived from the male germline. Therefore, we compared two different hepatic differentiation approaches and analyzed the generation of definitive endoderm progenitor cells and their further maturation into a hepatic phenotype using murine parthenogenetic ES cells, germline-derived pluripotent stem cells, and ES cells. Applying quantitative RT-PCR, both germline-derived pluripotent cell lines show similar differentiation capabilities as normal murine ES cells and can be considered an alternative source for pluripotent stem cells in regenerative medicine.

scholarly journals Full-term development of enucleated mouse oocytes fused with embryonic stem cells from different cell lines

Reproduction ◽  
2001 ◽  
pp. 729-733 ◽  
Author(s):  
T Amano ◽  
Y Kato ◽  
Y Tsunoda
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Clear Correlation ◽  
Developmental Potential ◽  
Mouse Oocytes

The developmental potential of enucleated mouse oocytes receiving embryonic stem cells from ten lines with either the same or different genetic backgrounds using the cell fusion method was examined in vitro and in vivo. The development of nuclear-transferred oocytes into blastocysts was high (34-88%). However, there was no clear correlation between development into blastocysts after nuclear transfer and the chimaera formation rate of embryonic stem cells. The development into live young was low (1-3%) in all cell lines and 14 of 19 young died shortly after birth. Most of the live young had morphological abnormalities. Of the five remaining mice, two died at days 23 and 30 after birth, but the other three mice are still active at days 359 (mouse 1) and 338 (mice 4 and 5) after birth, with normal fertility. However, the reasons for the abnormalities and postnatal death of embryonic stem cell-derived mice are unknown.

Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3568-3577
Author(s):  
Scott T. Magness ◽  
Antonio Tugores ◽  
David A. Brenner
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Translational Control ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Egfp Expression ◽  
Cell Stage ◽  
Cis Element

Ferrochelatase, the last enzyme in the heme pathway, chelates protoporphyrin IX and iron to form heme and is mutated in protoporphyria. The ferrochelatase gene is expressed in all tissues at low levels to provide heme for essential heme-containing proteins and is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF–E2 sites, all of which bind their cognatetrans-acting factors in vitro. To investigate the role of these elements during erythropoiesis, we introduced expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells. EGFP expression was monitored during hematopoietic differentiation in vitro using flow cytometry. We show that a promoter fragment containing the Sp1 sites, the NF–E2 and GATA elements, was sufficient to confer developmental-specific expression of the EGFP transgene, with an expression profile identical to that of the endogenous gene. In this system the −0.275 kb NF–E2 cis-element is required for erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between −0.375kb and −1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria. There was no effect of the G haplotype on transcriptional activity of the −1.1 kb transgene.

Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors

Blood ◽  
2008 ◽  
Vol 111 (11) ◽  
pp. 5298-5306 ◽  
Author(s):  
Naoya Takayama ◽  
Hidekazu Nishikii ◽  
Joichi Usui ◽  
Hiroko Tsukui ◽  
Akira Sawaguchi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Efficient Production ◽  
Hematopoietic Progenitors ◽  
Alternative Source ◽  
Promising Tool ◽  
Spherical Cells

Abstract Human embryonic stem cells (hESCs) could potentially represent an alternative source for blood transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. When we cultured hESCs on either C3H10T1/2 or OP-9 cells to facilitate hematopoiesis, we found that exogenous administration of vascular endothelial growth factor promoted the emergence of sac-like structures, which we named embryonic stem cell–derived sacs (ES-sacs). These ES-sacs consisted of multiple cysts demarcated by cellular monolayers that retained some of the properties of endothelial cells. The spherical cells inside ES-sacs expressed primarily CD34, along with VE-cadherin, CD31, CD41a, and CD45, and were able to form hematopoietic colonies in semisolid culture and to differentiate into mature megakaryocytes by day 24 in the presence of thrombopoietin. Apparently, ES-sacs provide a suitable environment for hematopoietic progenitors. Relatively large numbers of mature megakaryocytes could be induced from the hematopoietic progenitors within ES-sacs, which were then able to release platelets that displayed integrin αIIbβ3 activation and spreading in response to ADP or thrombin. This novel protocol thus provides a means of generating platelets from hESCs, which could serve as the basis for efficient production of platelets for clinical transfusion and studies of thrombopoiesis.

scholarly journals Erythropoiesis In Vitro—A Research and Therapeutic Tool in Thalassemia

2019 ◽  
Vol 8 (12) ◽  
pp. 2124 ◽  
Author(s):  
Eitan Fibach
Keyword(s):  
Stem Cells ◽  
Clinical Application ◽  
Financial Burden ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Alternative Source ◽  
Erythroid Progenitor ◽  
Globin Chains ◽  
Induced Pluripotent

Thalassemia (thal) is a hereditary chronic hemolytic anemia due to a partial or complete deficiency in the production of globin chains, in most cases, α or β, which compose, together with the iron-containing porphyrins (hemes), the hemoglobin molecules in red blood cells (RBC). The major clinical symptom of β-thal is severe chronic anemia—a decrease in RBC number and their hemoglobin content. In spite of the improvement in therapy, thal still severely affects the quality of life of the patients and their families and imposes a substantial financial burden on the community. These considerations position β-thal, among other hemoglobinopathies, as a major health and social problem that deserves increased efforts in research and its clinical application. These efforts are based on clinical studies, experiments in animal models and the use of erythroid cells grown in culture. The latter include immortal cell lines and cultures initiated by erythroid progenitor and stem cells derived from the blood and RBC producing (erythropoietic) sites of normal and thal donors, embryonic stem cells, and recently, "induced pluripotent stem cells" generated by manipulation of differentiated somatic cells. The present review summarizes the use of erythroid cultures, their technological aspects and their contribution to the research and its clinical application in thal. The former includes deciphering of the normal and pathological biology of the erythroid cell development, and the latter—their role in developing innovative therapeutics—drugs and methods of gene therapy, as well as providing an alternative source of RBC that may complement or substitute blood transfusions.

scholarly journals Cped1 promotes chicken SSCs formation with the aid of histone acetylation and transcription factor Sox2

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Chen Zhang ◽  
Fei Wang ◽  
Qisheng Zuo ◽  
Changhua Sun ◽  
Jing Jin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Histone Acetylation ◽  
Histone Deacetylase Inhibitors ◽  
Embryonic Stem ◽  
Control Area ◽  
Luciferase Reporter ◽  
Marker Genes

Spermatogonial stem cells (SSCs) may apply to gene therapy, regenerative medicine in place of embryonic stem cells (ESCs). However, the application of SSCs was severely limited by the low induction efficiency and the lack of thorough analysis of the regulatory mechanisms of SSCs formation. Current evidences have demonstrated multiple marker genes of germ cells, while genes that specifically regulate the formation of SSCs have not been explored. In our study, cadherin-like and PC-esterase domain containing 1 (Cped1) expressed specifically in SSCs based on RNA-seq data analysis. To study the function of Cped1 in the formation of SSCs, we successfully established a CRISPR/Cas9 knockout system. The gene disruption frequency is 37% in DF1 and 25% in ESCs without off-target effects. Knockout of Cped1 could significantly inhibit the formation of SSCs in vivo and in vitro. The fragment of −1050 to −1 bp had the activity as Cped1 gene promoter. Histone acetylation could regulate the expression of Cped1. We added 5-azaeytidi (DNA methylation inhibitors) and TSA (histone deacetylase inhibitors) respectively during the cultivation of SSCs. TSA was validated to promote the transcription of Cped1. Dual-luciferase reporter assay revealed that active control area of the chicken Cped1 gene is −296 to −1 bp. There are Cebpb, Sp1, and Sox2 transcription factor binding sites in this region. Point-mutation experiment results showed that Sox2 negatively regulates the transcription of Cped1. Above results demonstrated that Cped1 is a key gene that regulates the formation of SSCs. Histone acetylation and transcription factor Sox2 participate in the regulation of Cped1.

311 EXPRESSION OF PLURIPOTENT MARKER NUCLEOSTEMIN IN BUFFALO (BUBALUS BUBALIS) EMBRYOS AND EMBRYONIC STEM CELLS GENERATED THROUGH PARTHENOGENETIC ACTIVATION

2011 ◽  
Vol 23 (1) ◽  
pp. 252
Author(s):  
K. P. Singh ◽  
R. Kaushik ◽  
R. Sharma ◽  
S. Kala ◽  
A. George ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
In Vitro Maturation ◽  
Developmental Stages ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Stage ◽  
Parthenogenetic Activation ◽  
Immature Oocytes

Nucleostemin is a newly found putative GTPase protein that binds to P53 and exists mainly in the nucleoli and at a very low level in nucleoplasm of undifferentiated embryonic stem cells (ESC) and myeloid stem cells but is not expressed in committed and terminally differentiated cells. Embryonic stem cells are pluripotent cells derived from the inner cell mass (ICM) of blastocysts. The ICM and ESC express a number of transcription factors, and their expression is used as a pluripotency marker in the ESC of many species. The present study was undertaken to identify expression of the nucleostemin gene in different developmental stages of buffalo embryos and cultured ESC. Parthenogenetic activation is a process by which an oocyte can be developed up to blastocyst without fertilization. The parthenotes were produced by following protocol. Briefly, immature oocytes were aspirated from slaughterhouse buffalo ovaries and subjected to in vitro maturation for 24 h in a CO2 incubator (5% O2, 5% CO2, 90–95% relative humidity) at 38.5°C. After 24 h of in vitro maturation, oocytes were activated by exposure to 7% ethanol for 7 min, followed by incubation with 2 mM 6-dimethyl aminopurine in CR2 medium for 3.5 h, and they were then subjected to in vitro culture. The activated embryos were cultured for 8 days in CR2 medium containing 0.6% BSA and 10% FBS to obtain different stages (immature and mature oocytes 2-, 4-,8–16-cell, morula, and blastocyst) of embryos. A total of 23 blastocysts were produced parthenogenetically, of which 5 blastocysts were used for nucleostemin expression and the rest were used for ICM isolation. The isolated ICM were subsequently cultured on mitomycin-C (10 μg mL–1) treated buffalo fetal fibroblast feeder layer in DMEM medium supplemented with 20% fetal bovine serum, 1 000 IU mL–1 of mouse leukemia inhibitory factor, 1% nonessential amino acids, 2 mM L-glutamine, and 50 μg mL–1 gentamycin. These ESC were cultured up to 5 passages. The 5 embryos of different developmental stages and a clump of ESC were used for nucleostemin expression. The total RNA was isolated and transcribed using Cell-to-cDNA-II (Ambion, Austin, TX, USA) according to manufacturer protocol. To amplify the nucleostemin gene, the PCR cycle was carried out and included heating to 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s. The expressions of nucleostemin transcript were observed in all the developmental stages including immature and mature oocytes. The transcript was highly expressed in the 2-cell stage, blastocysts, and ESC, but immature oocytes and 8–16-cell stage showed lower expression. The experiment was repeated, and the same result was found. To our knowledge this is the first report in buffalo. It is concluded that the transcript was expressed in all the early stages of parthenogenetically derived buffalo embryos from immature oocytes to blastocysts and continued to be expressed in ESC. This work was funded by NAIP, C-420678075, India.

Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3568-3577 ◽  
Author(s):  
Scott T. Magness ◽  
Antonio Tugores ◽  
David A. Brenner
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Translational Control ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Egfp Expression ◽  
Cell Stage ◽  
Cis Element

Abstract Ferrochelatase, the last enzyme in the heme pathway, chelates protoporphyrin IX and iron to form heme and is mutated in protoporphyria. The ferrochelatase gene is expressed in all tissues at low levels to provide heme for essential heme-containing proteins and is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF–E2 sites, all of which bind their cognatetrans-acting factors in vitro. To investigate the role of these elements during erythropoiesis, we introduced expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells. EGFP expression was monitored during hematopoietic differentiation in vitro using flow cytometry. We show that a promoter fragment containing the Sp1 sites, the NF–E2 and GATA elements, was sufficient to confer developmental-specific expression of the EGFP transgene, with an expression profile identical to that of the endogenous gene. In this system the −0.275 kb NF–E2 cis-element is required for erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between −0.375kb and −1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria. There was no effect of the G haplotype on transcriptional activity of the −1.1 kb transgene.

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