OBJECTIVE: To investigate an method for hepatic differentiation from embryonic stem cells (ES cells) in vitro and the resulting differentiation ratio, in order to develop a procedure for producing a new type of hepato...OBJECTIVE: To investigate an method for hepatic differentiation from embryonic stem cells (ES cells) in vitro and the resulting differentiation ratio, in order to develop a procedure for producing a new type of hepatocyte for hepatocyte replacement therapy in the treatment of liver failure. METHODS: ES cells from Balb/C mice were cultured and maintained in an undifferentiated state in gelatin-coated dishes using Dulbecco's modified Eagle's medium (DMEM) containing 1000 U/ml leukemia inhibitory factor (LIF). Then, LIF was withdrawn from the DMEM to allow the ES cells to develop into embryonic bodies (EBs). EBs were plated onto tissue culture dishes, and growth factors such as acidicfibroblast growth factor (aFGF) and hepatocyte growth factor (HGF) were added to the medium to promote directional differentiation. The course of development and differentiation was observed dynamically using an inversion microscope. The expression of hepatic proteins, such as alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 8 (CK8), cytokeratin 18 (CK18), in cytoplasm was analyzed by immunocytochemistry (ICC). The concentration of ALB in the medium was determined dynamically by radioimmunoassay (RIA). RESULTS: ES cells replicated as clones, without differentiating, in DMEM containing LIF. They developed into EBs in medium without LIF. Our ICC assay showed that differentiating cells did not express hepatic proteins, such as AFP, ALB, CK8, and CK18 until day 7, day 9, day 11, and day 11, respectively (up to 2 days later when growth factors are not present). The concentration of AFP in the medium was first detected on day 8, at a concentration of 3.4 ng/ml, and increased to 22.8 ng/ml by day 15. The concentration of ALB in the medium was 0.2 micro g/ml on day 11, and increased to 2.2 micro g/ml by day 15. ALB-positive cells under ICC manifest morphological structures were consistent with normal mouse hepatocytes. The differentiation ratio of hepatocytes in the ES cell differentiation system was 30% on day 15 (significantly lower withou展开更多
One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which 2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos, which are regarded as a...One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which 2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos, which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is often assumed that the block of early development is due to the failure of/ygotic gene activation (ZGA) in cultured embryos. In this study we examined protein synthesis patterns by two-dimensional gel eleetrophoresis of [35 S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strain were compared in their development both in vitro and in vivo. The detection of TRC expression, a marker of ZGA. at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even in the 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as compared with normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG, TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo. But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidences suggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to the disability in initiating ZGA. but to a delay of ZGA.展开更多
During embryo development, the vascular precursors and ground tissue stem cells divide to renew them-selves and produce the vascular tissue, endodermal cells, and cortical cells. However, the molecular mech-anisms reg...During embryo development, the vascular precursors and ground tissue stem cells divide to renew them-selves and produce the vascular tissue, endodermal cells, and cortical cells. However, the molecular mech-anisms regulating division of these stem cells have remained largely elusive. In this study, we show that loss of function of SOMATIC EMBRYOGENESIS RECEPTOR-UKE KINASE (SERK) genes results in aberrant em-bryo development. Fewer cortical, endodermal, and vascular cells are generated in the embryos of serk1 serk2bak1 triple mutants. WUSCHEL-RELATED HOMBOBOX5 (WOXS) is ectopically expressed in vascular cells of serkl serk2 bak1 embryos. The first transverse division of vascular precursors in mid-globular em-bryos and second asymmetric division of ground tissue stem cells in early-heart embryos are abnormally altered to a longitudinal division. The embryo defects can be partially rescued by constitutively activated mitogen-activated protein kinase (MAPK) kinase kinase YODA (YDA) and MAPK kinase MKK5. Taken together, our results reveal that SERK-mediated signals regulate division patterns of vascular precursors and ground tissue stem cells, likely via the YDA-MKK4/5 cascade, during embryo development.展开更多
Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before poll...Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and primary endosperm cytoplasm during fertilization.展开更多
基金ThisworkwassupportedbytheNationalKeyResearchFoundationofChina ( 973 ) (No 2 0 0 1CB5 10 10 1)andtheNationalNaturalScienceFoundationofChina (No 3 0 2 3 0 3 5 0 No 60 2 780 14 )
文摘OBJECTIVE: To investigate an method for hepatic differentiation from embryonic stem cells (ES cells) in vitro and the resulting differentiation ratio, in order to develop a procedure for producing a new type of hepatocyte for hepatocyte replacement therapy in the treatment of liver failure. METHODS: ES cells from Balb/C mice were cultured and maintained in an undifferentiated state in gelatin-coated dishes using Dulbecco's modified Eagle's medium (DMEM) containing 1000 U/ml leukemia inhibitory factor (LIF). Then, LIF was withdrawn from the DMEM to allow the ES cells to develop into embryonic bodies (EBs). EBs were plated onto tissue culture dishes, and growth factors such as acidicfibroblast growth factor (aFGF) and hepatocyte growth factor (HGF) were added to the medium to promote directional differentiation. The course of development and differentiation was observed dynamically using an inversion microscope. The expression of hepatic proteins, such as alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 8 (CK8), cytokeratin 18 (CK18), in cytoplasm was analyzed by immunocytochemistry (ICC). The concentration of ALB in the medium was determined dynamically by radioimmunoassay (RIA). RESULTS: ES cells replicated as clones, without differentiating, in DMEM containing LIF. They developed into EBs in medium without LIF. Our ICC assay showed that differentiating cells did not express hepatic proteins, such as AFP, ALB, CK8, and CK18 until day 7, day 9, day 11, and day 11, respectively (up to 2 days later when growth factors are not present). The concentration of AFP in the medium was first detected on day 8, at a concentration of 3.4 ng/ml, and increased to 22.8 ng/ml by day 15. The concentration of ALB in the medium was 0.2 micro g/ml on day 11, and increased to 2.2 micro g/ml by day 15. ALB-positive cells under ICC manifest morphological structures were consistent with normal mouse hepatocytes. The differentiation ratio of hepatocytes in the ES cell differentiation system was 30% on day 15 (significantly lower withou
基金supported by grants from Shanghai Scientific Development Foundation(No.01JC14047)National Key Basic Research Project“973”(No.G199905592).
文摘One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which 2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos, which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is often assumed that the block of early development is due to the failure of/ygotic gene activation (ZGA) in cultured embryos. In this study we examined protein synthesis patterns by two-dimensional gel eleetrophoresis of [35 S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strain were compared in their development both in vitro and in vivo. The detection of TRC expression, a marker of ZGA. at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even in the 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as compared with normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG, TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo. But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidences suggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to the disability in initiating ZGA. but to a delay of ZGA.
基金National Natural Science Foundation of China (31770312, 31530005, 31471402, 31720103902, 31270229, 31070283)the Ministry of Education (113058A. NCET-12-0249)+2 种基金the 111 Project (B16022)the Fundamental Research Funds for the Central Universities (lzujbky-2018-kb05)the Gansu Provincial Science & Technology Department (17ZD2NA015-06, 17ZD2NA016-5).
文摘During embryo development, the vascular precursors and ground tissue stem cells divide to renew them-selves and produce the vascular tissue, endodermal cells, and cortical cells. However, the molecular mech-anisms regulating division of these stem cells have remained largely elusive. In this study, we show that loss of function of SOMATIC EMBRYOGENESIS RECEPTOR-UKE KINASE (SERK) genes results in aberrant em-bryo development. Fewer cortical, endodermal, and vascular cells are generated in the embryos of serk1 serk2bak1 triple mutants. WUSCHEL-RELATED HOMBOBOX5 (WOXS) is ectopically expressed in vascular cells of serkl serk2 bak1 embryos. The first transverse division of vascular precursors in mid-globular em-bryos and second asymmetric division of ground tissue stem cells in early-heart embryos are abnormally altered to a longitudinal division. The embryo defects can be partially rescued by constitutively activated mitogen-activated protein kinase (MAPK) kinase kinase YODA (YDA) and MAPK kinase MKK5. Taken together, our results reveal that SERK-mediated signals regulate division patterns of vascular precursors and ground tissue stem cells, likely via the YDA-MKK4/5 cascade, during embryo development.
基金This research was supported by a RGC grant from the Research Grants Council of Hong Kong anda CRCG grant from University of Hong Kong to B. Q. H, a CRCG grant to S. Y. Z, NSF grant No. MCB-9601087 to P. K. H., and the National Key Basic Research Projec
文摘Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and primary endosperm cytoplasm during fertilization.
