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Manabu Ozawa - One of the best experts on this subject based on the ideXlab platform.

  • development of fgf2 dependent pluripotent stem Cells showing naive state characteristics from murine preimplantation Inner Cell Mass
    Stem Cell Research, 2014
    Co-Authors: Manabu Ozawa, Eri Kawakami, Reiko Sakamoto, Takayuki Shibasaki, Akiteru Goto, Nobuaki Yoshida
    Abstract:

    Two distinct types of embryonic pluripotent stem Cells can be established from either the Inner Cell Mass (ICM) of preimplantation blastocyst (leukemia inhibitory factor (LIF)-dependent embryonic stem Cell, ESC, called naive state) or the epiblast of postimplantation fetuses (fibroblast growth factor 2 (FGF2)-dependent epiblast stem Cells, EpiSC, called primed state). Here, we report that naive pluripotent stem Cell was established from the ICM, but maintained its self-renewal by treatment with FGF2 and mouse embryonic fibroblasts (MEFs) when they were exposed FGF2 during establishment. This Cell line is competent to contribute to chimeric animals, including germ Cells, at high efficiency. The ERK1/2, SMAD2/3, and JAK/STAT3 pathways are essential to maintain self-renewal. Inhibition of ERK1/2 or SMAD2/3 initiates transition to a naive state ESC-like state, whereas inhibition of JAK/STAT3 promotes a primed EpiSC-like character. Our present results could provide novel insights into understanding the growth factor environment and ICM plasticity, and mechanisms which orchestrate the pluripotency of embryonic stem Cells and the capacity for chimeric contributions.

  • regulation of pluripotency of Inner Cell Mass and growth and differentiation of trophectoderm of the bovine embryo by colony stimulating factor 2
    Biology of Reproduction, 2013
    Co-Authors: Kyle B. Dobbs, Manabu Ozawa, Miki Sakatani, Alan D Ealy, F A Khan, J I Moss, P J Hansen
    Abstract:

    Colony-stimulating factor 2 (CSF2) enhances competence of the bovine embryo to establish and maintain pregnancy after the embryo is transferred into a recipient. Mechanisms involved could include regulation of lineage commitment, growth, or differentiation of the Inner Cell Mass (ICM) and trophectoderm (TE). Experiments were conducted to evaluate regulation by CSF2 of pluripotency of the ICM and differentiation and growth of the TE. Embryos were cultured with 10 ng/ml recombinant bovine CSF2 or a vehicle control from Days 5 to 7 or 6 to 8 postinsemination. CSF2 increased the number of putative zygotes that developed to blastocysts when the percent of embryos becoming blastocysts in the control group was low but decreased blastocyst yield when blastocyst development in controls was high. ICM isolated from blastocysts by lysing the trophectoderm using antibody and complement via immunosurgery were more likely to survive passage when cultured on mitomycin C-treated fetal fibroblasts if derived from blastocysts treated with CSF2 than if from control blastocysts. There was little effect of CSF2 on characteristics of TE outgrowths from blastocysts. The exception was a decrease in outgrowth size for embryos treated with CSF2 from Days 5 to 7 and an increase in expression of CDX2 when treatment was from Days 6 to 8. Expression of the receptor subunit gene CSF2RA increased from the zygote stage to the 9–16 Cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages. In conclusion, CSF2 improves competence of the ICM to survive in a pluripotent state and alters TE outgrowths. Actions of CSF2 occur through a signaling pathway that is likely to be independent of CSF2RB. bovine, CSF2 receptor, colony stimulating factor 2, embryo, Inner Cell Mass, trophectoderm

  • Global gene expression of the Inner Cell Mass and trophectoderm of the bovine blastocyst
    BMC Developmental Biology, 2012
    Co-Authors: Manabu Ozawa, Miki Sakatani, Kyle B. Dobbs, Savita Shanker, Fahong Yu, Rui Yamashita, Shunichi Wakabayashi, Kenta Nakai, Mateus José Sudano, William G Farmerie
    Abstract:

    Background The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent Inner Cell Mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated Cell sorting, and cDNA sequenced using the SOLiD 4.0 system. Results A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG , SOX2 , and STAT3 ) and TE ( ELF5 , GATA3 , and KRT18 ) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human. Conclusion Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two Cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in Cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.

  • Importance of culture conditions during the morula-to-blastocyst period on capacity of Inner Cell-Mass Cells of bovine blastocysts for establishment of self-renewing pluripotent Cells.
    Theriogenology, 2012
    Co-Authors: Manabu Ozawa, Miki Sakatani, Katherine E. Hankowski, Kyle B. Dobbs, Naohiro Terada, Peter J. Hansen
    Abstract:

    The hypothesis was tested that the pluripotency of the Inner Cell Mass (ICM) of the bovine embryo is enhanced by the glycogen synthase kinase-3β inhibitor CHIR99021 and the MAPK1 and MAPK3 inhibitor PD032591. Treatment with the two inhibitors from Days 6 to 8 after insemination increased blastocyst steady state concentrations of mRNA for NANOG (P < 0.05) and SOX2 (P = 0.055) and tended to decrease (P = 0.09) expression of GATA6. To evaluate pluripotency, the Inner Cell Mass was isolated by immunosurgery at Day 8, seeded on a feeder layer of bovine embryonic fibroblasts, and cultured in the presence of the inhibitors. Ten of 52 (19%) ICM from control embryos had primary outgrowth formation vs. 23 of 50 (46%) of the ICM from embryos cultured with inhibitors (P < 0.01). For ICM outgrowths from embryos cultured without inhibitors, colonies either did not persist through Passage 2 or became differentiated. In contrast, for the inhibitor group, four colonies survived beyond Passage 2, and one line persisted for 19 passages. This Cell line possessed alkaline phosphatase activity, expressed several genes characteristically expressed in pluripotent Cells, and differentiated into embryoid bodies when cultured in the absence of the signal transduction inhibitors and the feeder layer. Propagation of the Cells was difficult due to slow growth and inefficiency in survival through each passage. In conclusion, exposure to inhibitors during the morula-blastocyst transition facilitated formation of self-renewing pluripotent Cell lines from bovine blastocysts.

  • a novel method for purification of Inner Cell Mass and trophectoderm Cells from blastocysts using magnetic activated Cell sorting
    Fertility and Sterility, 2011
    Co-Authors: Manabu Ozawa, Peter J. Hansen
    Abstract:

    Objective To develop a simple method to purify blastomeres of Inner Cell Mass (ICM) and trophectoderm (TE) lineage using magnetic activated Cell sorting. Design Prospective laboratory study. Setting Embryology research laboratory. Patient(s) None. Intervention(s) Trophectoderm Cells of zona-free blastocysts were labeled with concanavalin A conjugated to FITC, and every nucleus in the blastocyst was labeled with Hoechst 33342. The labeled blastocyst was disaggregated to single Cells by trypsin treatment followed by pipetting using a finely drawn, flame-polished micropipet. Disaggregated blastomeres were incubated with anti-FITC antibody conjugated to magnetic microbeads and subjected to magnetic Cell sorting to separate Cells into FITC-positive and -negative fractions. Main Outcome Measure(s) Purity and gene expression. Result(s) In the FITC-positive fraction, an average of 91.2% of Cells was dual-labeled with FITC and Hoechst, whereas only 7.8% of FITC negative fractions were labeled with FITC. Expression of CDX2, a trophectoderm marker, was significantly higher in the FITC-positive fraction, whereas expression of NANOG, an Inner Cell Mass marker, was significantly higher in the FITC-negative fraction. Conclusion(s) Highly purified trophectoderm Cells or Inner Cell Mass Cells can be collected using magnetic activated Cell sorting. This method can be useful for understanding differentiation and function of Cell lineages in the blastocyst.

Peter J. Hansen - One of the best experts on this subject based on the ideXlab platform.

  • Role of chemokine (C-C motif) ligand 24 in spatial arrangement of the Inner Cell Mass of the bovine embryo.
    Biology of reproduction, 2017
    Co-Authors: Verónica M. Negrón-pérez, Dorianmarie Vargas-franco, Peter J. Hansen
    Abstract:

    The process of spatial rearrangement of Cells of the Inner Cell Mass (ICM) that are destined to become hypoblast is not well understood. The observation that the chemokine (C-C motif) ligand 24 (CCL24) and several other genes involved in chemokine signaling are expressed more in the ICM than in the trophectoderm of the bovine embryo resulted in the hypothesis that CCL24 participates in spatial organization of the ICM. Temporally, expression of CCL24 in the bovine embryo occurs coincidently with blastocyst formation: transcript abundance was low until the late morula stage, peaked in the blastocyst at Day 7 of development and declined by Day 9. Treatment of embryos with two separate antagonists of C-C motif chemokine receptor 3 (the prototypical receptor for CCL24) decreased the percent of GATA6+ Cells (hypoblast precursors) that were located in the outside of the ICM. Similarly, injection of zygotes with a CCL24-specific morpholino decreased the percent of GATA6+ Cells in the outside of the ICM. In conclusion, CCL24 assists in spatial arrangement of the ICM in the bovine embryo. This experiment points to new functions of chemokine signaling in the bovine embryo and is consistent with the idea that Cell migration is involved in the spatial organization of hypoblast Cells in the blastocyst.

  • Importance of culture conditions during the morula-to-blastocyst period on capacity of Inner Cell-Mass Cells of bovine blastocysts for establishment of self-renewing pluripotent Cells.
    Theriogenology, 2012
    Co-Authors: Manabu Ozawa, Miki Sakatani, Katherine E. Hankowski, Kyle B. Dobbs, Naohiro Terada, Peter J. Hansen
    Abstract:

    The hypothesis was tested that the pluripotency of the Inner Cell Mass (ICM) of the bovine embryo is enhanced by the glycogen synthase kinase-3β inhibitor CHIR99021 and the MAPK1 and MAPK3 inhibitor PD032591. Treatment with the two inhibitors from Days 6 to 8 after insemination increased blastocyst steady state concentrations of mRNA for NANOG (P < 0.05) and SOX2 (P = 0.055) and tended to decrease (P = 0.09) expression of GATA6. To evaluate pluripotency, the Inner Cell Mass was isolated by immunosurgery at Day 8, seeded on a feeder layer of bovine embryonic fibroblasts, and cultured in the presence of the inhibitors. Ten of 52 (19%) ICM from control embryos had primary outgrowth formation vs. 23 of 50 (46%) of the ICM from embryos cultured with inhibitors (P < 0.01). For ICM outgrowths from embryos cultured without inhibitors, colonies either did not persist through Passage 2 or became differentiated. In contrast, for the inhibitor group, four colonies survived beyond Passage 2, and one line persisted for 19 passages. This Cell line possessed alkaline phosphatase activity, expressed several genes characteristically expressed in pluripotent Cells, and differentiated into embryoid bodies when cultured in the absence of the signal transduction inhibitors and the feeder layer. Propagation of the Cells was difficult due to slow growth and inefficiency in survival through each passage. In conclusion, exposure to inhibitors during the morula-blastocyst transition facilitated formation of self-renewing pluripotent Cell lines from bovine blastocysts.

  • a novel method for purification of Inner Cell Mass and trophectoderm Cells from blastocysts using magnetic activated Cell sorting
    Fertility and Sterility, 2011
    Co-Authors: Manabu Ozawa, Peter J. Hansen
    Abstract:

    Objective To develop a simple method to purify blastomeres of Inner Cell Mass (ICM) and trophectoderm (TE) lineage using magnetic activated Cell sorting. Design Prospective laboratory study. Setting Embryology research laboratory. Patient(s) None. Intervention(s) Trophectoderm Cells of zona-free blastocysts were labeled with concanavalin A conjugated to FITC, and every nucleus in the blastocyst was labeled with Hoechst 33342. The labeled blastocyst was disaggregated to single Cells by trypsin treatment followed by pipetting using a finely drawn, flame-polished micropipet. Disaggregated blastomeres were incubated with anti-FITC antibody conjugated to magnetic microbeads and subjected to magnetic Cell sorting to separate Cells into FITC-positive and -negative fractions. Main Outcome Measure(s) Purity and gene expression. Result(s) In the FITC-positive fraction, an average of 91.2% of Cells was dual-labeled with FITC and Hoechst, whereas only 7.8% of FITC negative fractions were labeled with FITC. Expression of CDX2, a trophectoderm marker, was significantly higher in the FITC-positive fraction, whereas expression of NANOG, an Inner Cell Mass marker, was significantly higher in the FITC-negative fraction. Conclusion(s) Highly purified trophectoderm Cells or Inner Cell Mass Cells can be collected using magnetic activated Cell sorting. This method can be useful for understanding differentiation and function of Cell lineages in the blastocyst.

  • A novel method for purification of Inner Cell Mass and trophectoderm Cells from blastocysts using magnetic activated Cell sorting
    Fertility and Sterility, 2010
    Co-Authors: Manabu Ozawa, Peter J. Hansen
    Abstract:

    Objective: To develop a simple method to purify blastomeres of Inner Cell Mass (ICM) and trophectoderm (TE) lineage using magnetic activated Cell sorting. Design: Prospective laboratory study. Setting: Embryology research laboratory. Patient(s): None. Intervention(s): Trophectoderm Cells of zona-free blastocysts were labeled with concanavalin A conjugated to FITC, and every nucleus in the blastocyst was labeled with Hoechst 33342. The labeled blastocyst was disaggregated to single Cells by trypsin treatment followed by pipetting using a finely drawn, flame-polished micropipet. Disaggregated blastomeres were incubated with anti-FITC antibody conjugated to magnetic microbeads and subjected to magnetic Cell sorting to separate Cells into FITC-positive and -negative fractions. Main Outcome Measure(s): Purity and gene expression. Result(s): In the FITC-positive fraction, an average of 91.2% of Cells was dual-labeled with FITC and Hoechst, whereas only 7.8% of FITC negative fractions were labeled with FITC. Expression of CDX2, a trophectoderm marker, was significantly higher in the FITC-positive fraction, whereas expression of NANOG, an Inner Cell Mass marker, was significantly higher in the FITC-negative fraction. Conclusion(s): Highly purified trophectoderm Cells or Inner Cell Mass Cells can be collected using magnetic activated Cell sorting. This method can be useful for understanding differentiation and function of Cell lineages in the blastocyst. (Fertil Steril� 2011;95:799‐802. � 2011 by American Society for Reproductive Medicine.)

Alan D Ealy - One of the best experts on this subject based on the ideXlab platform.

  • interleukin 6 requires jak to stimulate Inner Cell Mass expansion in bovine embryos
    Reproduction, 2019
    Co-Authors: Lydia K Wooldridge, Sally E Johnson, Rebecca R Cockrum, Alan D Ealy
    Abstract:

    Supplementing interleukin-6 (IL6) to in vitro-produced bovine embryos increases Inner Cell Mass (ICM) Cell numbers in blastocysts. A series of studies were completed to further dissect this effect. Treatment with IL6 increased ICM Cell numbers in early, regular and expanded blastocysts but had no effect on morulae total Cell number. Treatment with IL6 for 30 min induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation in all blastomeres in early morulae and specifically within the ICM in blastocysts. Also, IL6 supplementation increased SOCS3 mRNA abundance, a STAT3-responsive gene, in blastocysts. Chemical inhibition of Janus kinase (JAK) activity from day 5 to day 8 prevented STAT3 activation and the IL6-induced ICM Cell number increase. Global transcriptome analysis of blastocysts found that transcripts for IL6 and its receptor subunits (IL6R and IL6ST) were the most abundantly expressed IL6 family ligand and receptors. These results indicate that IL6 increases ICM Cell numbers as the ICM lineage emerges at the early blastocyst stage through a STAT3-dependent mechanism. Also, IL6 appears to be the primary IL6 cytokine family member utilized by bovine blastocysts to control ICM Cell numbers.

  • interleukin 6 increases Inner Cell Mass numbers in bovine embryos
    BMC Developmental Biology, 2019
    Co-Authors: Lydia K Wooldridge, Alan D Ealy
    Abstract:

    Work in other species suggests that interleukin-6 (IL6) promotes early embryo development. It was unclear whether IL6 serves as an embryokine in cultured bovine embryos. This work was undertaken to elucidate the role of IL6 during in vitro bovine embryo production. Transcripts for IL6 and its two cognate receptor subunits (IL6R, IL6ST) were confirmed in bovine embryos from the 1-Cell to blastocyst stages. Supplementing 100 ng/ml recombinant bovine IL6 to in vitro-produced bovine embryos at day 1, 3 or 5 increased (P < 0.05) Inner Cell Mass (ICM) Cell number and the ICM:trophectoderm (TE) ratio but not TE Cell number. No increase in ICM or TE Cell number was observed after supplementation of 1 or 10 ng/ml IL6 beginning at either day 1 or 5. Sequential supplementation with 100 ng/ml IL6 at both day 1 and 5 (for a total of 200 ng/ml IL6) increased (P < 0.05) ICM Cell number to a greater extent than supplementing IL6 at a single time period in one study but not a second study. Additionally, providing 200 ng/ml IL6 beginning at day 1 or 5 yielded no further increase on ICM Cell numbers when compared to supplementing with 100 ng/ml IL6. IL6 treatment had no effect on cleavage or blastocyst formation in group culture. However, IL6 supplementation increased cleavage and day 8 blastocyst formation when bovine embryos were cultured individually. These results implicate IL6 as an embryokine that specifically increases ICM Cell numbers in bovine embryos and facilitates bovine blastocyst development in embryos cultured individually.

  • regulation of pluripotency of Inner Cell Mass and growth and differentiation of trophectoderm of the bovine embryo by colony stimulating factor 2
    Biology of Reproduction, 2013
    Co-Authors: Kyle B. Dobbs, Manabu Ozawa, Miki Sakatani, Alan D Ealy, F A Khan, J I Moss, P J Hansen
    Abstract:

    Colony-stimulating factor 2 (CSF2) enhances competence of the bovine embryo to establish and maintain pregnancy after the embryo is transferred into a recipient. Mechanisms involved could include regulation of lineage commitment, growth, or differentiation of the Inner Cell Mass (ICM) and trophectoderm (TE). Experiments were conducted to evaluate regulation by CSF2 of pluripotency of the ICM and differentiation and growth of the TE. Embryos were cultured with 10 ng/ml recombinant bovine CSF2 or a vehicle control from Days 5 to 7 or 6 to 8 postinsemination. CSF2 increased the number of putative zygotes that developed to blastocysts when the percent of embryos becoming blastocysts in the control group was low but decreased blastocyst yield when blastocyst development in controls was high. ICM isolated from blastocysts by lysing the trophectoderm using antibody and complement via immunosurgery were more likely to survive passage when cultured on mitomycin C-treated fetal fibroblasts if derived from blastocysts treated with CSF2 than if from control blastocysts. There was little effect of CSF2 on characteristics of TE outgrowths from blastocysts. The exception was a decrease in outgrowth size for embryos treated with CSF2 from Days 5 to 7 and an increase in expression of CDX2 when treatment was from Days 6 to 8. Expression of the receptor subunit gene CSF2RA increased from the zygote stage to the 9–16 Cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages. In conclusion, CSF2 improves competence of the ICM to survive in a pluripotent state and alters TE outgrowths. Actions of CSF2 occur through a signaling pathway that is likely to be independent of CSF2RB. bovine, CSF2 receptor, colony stimulating factor 2, embryo, Inner Cell Mass, trophectoderm

Meri T Firpo - One of the best experts on this subject based on the ideXlab platform.

  • gene expression profiles of human Inner Cell Mass Cells and embryonic stem Cells
    Differentiation, 2009
    Co-Authors: Renee Reijo A Pera, Christopher Dejonge, Nancy L Bossert, Mylene W M Yao, Jean Yee Hwa Yang, Narges Bani Asadi, Wing Hung Wong, Connie C Wong, Meri T Firpo
    Abstract:

    Human embryonic stem Cell (hESC) lines are derived from the Inner Cell Mass (ICM) of preimplantation human blastocysts obtained on days 5-6 following fertilization. Based on their derivation, they were once thought to be the equivalent of the ICM. Recently, however, studies in mice reported the derivation of mouse embryonic stem Cell lines from the epiblast; these epiblast lines bear significant resemblance to human embryonic stem Cell lines in terms of culture, differentiation potential and gene expression. In this study, we compared gene expression in human ICM Cells isolated from the blastocyst and embryonic stem Cells. We demonstrate that expression profiles of ICM clusters from single embryos and hESC populations were highly reproducible. Moreover, comparison of global gene expression between individual ICM clusters and human embryonic stem Cells indicated that these two Cell types are significantly different in regards to gene expression, with fewer than one half of all genes expressed in both Cell types. Genes of the isolated human Inner Cell Mass that are upregulated and downregulated are involved in numerous Cellular pathways and processes; a subset of these genes may impart unique characteristics to hESCs such as proliferative and self-renewal properties.

K. Filimonow - One of the best experts on this subject based on the ideXlab platform.

  • plasticity of the Inner Cell Mass in mouse blastocyst is restricted by the activity of fgf mapk pathway
    Scientific Reports, 2017
    Co-Authors: M. Wigger, K. Kisielewska, K. Filimonow, M. Maleszewski, Berenika Plusa, A. Suwińska
    Abstract:

    In order to ensure successful development, Cells of the early mammalian embryo must differentiate to either trophectoderm (TE) or Inner Cell Mass (ICM), followed by epiblast (EPI) or primitive endoderm (PE) specification within the ICM. Here, we deciphered the mechanism that assures the correct order of these sequential Cell fate decisions. We revealed that TE-deprived ICMs derived from 32-Cell blastocysts are still able to reconstruct TE during in vitro culture, confirming totipotency of ICM Cells at this stage. ICMs isolated from more advanced blastocysts no longer retain totipotency, failing to form TE and generating PE on their surface. We demonstrated that the transition from full potency to lineage priming is prevented by inhibition of the FGF/MAPK signalling pathway. Moreover, we found that after this first restriction step, ICM Cells still retain fate flexibility, manifested by ability to convert their fate into an alternative lineage (PE towards EPI and vice versa), until peri-implantation stage.

  • Plasticity of the Inner Cell Mass in mouse blastocyst is restricted by the activity of FGF/MAPK pathway
    Nature Publishing Group, 2017
    Co-Authors: M. Wigger, K. Kisielewska, K. Filimonow, B. Plusa, M. Maleszewski, A. Suwińska
    Abstract:

    Abstract In order to ensure successful development, Cells of the early mammalian embryo must differentiate to either trophectoderm (TE) or Inner Cell Mass (ICM), followed by epiblast (EPI) or primitive endoderm (PE) specification within the ICM. Here, we deciphered the mechanism that assures the correct order of these sequential Cell fate decisions. We revealed that TE-deprived ICMs derived from 32-Cell blastocysts are still able to reconstruct TE during in vitro culture, confirming totipotency of ICM Cells at this stage. ICMs isolated from more advanced blastocysts no longer retain totipotency, failing to form TE and generating PE on their surface. We demonstrated that the transition from full potency to lineage priming is prevented by inhibition of the FGF/MAPK signalling pathway. Moreover, we found that after this first restriction step, ICM Cells still retain fate flexibility, manifested by ability to convert their fate into an alternative lineage (PE towards EPI and vice versa), until peri-implantation stage