BMPs regulate differentiation of a putative visceral endoderm layer within human embryonic stem-cell-derived embryoid bodies

2007 ◽  
Vol 85 (1) ◽  
pp. 121-132 ◽  
Author(s):  
Brock J Conley ◽  
Sarah Ellis ◽  
Lerna Gulluyan ◽  
Richard Mollard
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Visceral Endoderm ◽  
Transmission Electron ◽  
Spherical Structures ◽  
Bmp Signalling ◽  
Serum Free Conditions

Human embryonic stem cells (HESCs), pluripotent cells derived from the inner cell mass (ICM) of human blastocysts, represent a novel tool for the study of early human developmental events. When cultured in suspension with serum, HESCs form spherical structures resembling embryoid bodies (EBs). We show that differentiation of HESCs within EBs occurs radially, with central cells then undergoing apoptosis in association with EB cavitation. Cells within the outer layer of cavitating EBs display stage-specific immunoreactivity to pan-keratin, cytokeratin-8, GATA6, α-fetoprotein, and transthyretin specific antibodies, and hybridization to disabled-2, GATA4, and GATA6 specific riboprobes. Transmission electron microscopy of these cells reveals clathrin-coated micropinocytotic vesicles, microvilli, and many vacuoles, a phenotype consistent with mouse visceral endoderm (VE) rather than mouse definitive or parietal endoderm. When cultured in media supplemented with the BMP inhibitor noggin, or in the absence of serum, HESC derivatives do not develop the mouse VE-like phenotype. The addition of BMP-4 to noggin-treated HESCs cultured in serum or in serum-free conditions reconstituted development of the VE-like phenotype. These data demonstrate that human EBs undergo developmental events similar to those of mouse EBs and that in vitro BMP signalling induces derivatives of the human ICM to express a phenotype similar to mouse VE.

scholarly journals Generation of embryonic stem cell lines from mouse blastocysts developed in vivo and in vitro: relation to Oct-4 expression

Reproduction ◽  
2006 ◽  
Vol 132 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S Tielens ◽  
B Verhasselt ◽  
J Liu ◽  
M Dhont ◽  
J Van Der Elst ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Stem Cell Lines

Embryonic stem (ES) cells are the source of all embryonic germ layer tissues. Oct-4 is essential for their pluripotency. Sincein vitroculture may influence Oct-4 expression, we investigated to what extent blastocysts culturedin vitrofrom the zygote stage are capable of expressing Oct-4 and generating ES cell lines. We comparedin vivowithin vitroderived blastocysts from B6D2 mice with regard to Oct-4 expression in inner cell mass (ICM) outgrowths and blastocysts. ES cells were characterized by immunostaining for alkaline phosphatase (ALP), stage-specific embryonic antigen-1 (SSEA-1) and Oct-4. Embryoid bodies were made to evaluate the ES cells’ differentiation potential. ICM outgrowths were immunostained for Oct-4 after 6 days in culture. A quantitative real-time PCR assay was performed on individual blastocysts. Of thein vitroderived blastocysts, 17% gave rise to ES cells vs 38% of thein vivoblastocysts. Six-day old outgrowths fromin vivodeveloped blastocysts expressed Oct-4 in 55% of the cases vs 31% of thein vitroderived blastocysts. The amount of Oct-4 mRNA was significantly higher for freshly collectedin vivoblastocysts compared toin vitrocultured blastocysts.In vitrocultured mouse blastocysts retain the capacity to express Oct-4 and to generate ES cells, be it to a lower level thanin vivoblastocysts.

scholarly journals Absence of Basement Membranes after Targeting the LAMC1 Gene Results in Embryonic Lethality Due to Failure of Endoderm Differentiation

1999 ◽  
Vol 144 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Neil Smyth ◽  
H. Seda Vatansever ◽  
Patricia Murray ◽  
Michael Meyer ◽  
Christian Frie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Basement Membrane ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Basement Membranes ◽  
Embryoid Bodies ◽  
Endoderm Differentiation

The LAMC1 gene coding for the laminin γ1 subunit was targeted by homologous recombination in mouse embryonic stem cells. Mice heterozygous for the mutation had a normal phenotype and were fertile, whereas homozygous mutant embryos did not survive beyond day 5.5 post coitum. These embryos lacked basement membranes and although the blastocysts had expanded, primitive endoderm cells remained in the inner cell mass, and the parietal yolk sac did not develop. Cultured embryonic stem cells appeared normal after targeting both LAMC1 genes, but the embryoid bodies derived from them also lacked basement membranes, having disorganized extracellular deposits of the basement membrane proteins collagen IV and perlecan, and the cells failed to differentiate into stable myotubes. Secretion of the linking protein nidogen and a truncated laminin α1 subunit did occur, but these were not deposited in the extracellular matrix. These results show that the laminin γ1 subunit is necessary for laminin assembly and that laminin is in turn essential for the organization of other basement membrane components in vivo and in vitro. Surprisingly, basement membranes are not necessary for the formation of the first epithelium to develop during embryogenesis, but first become required for extra embryonic endoderm differentiation.

195 DEVELOPMENT OF INNER CELL MASS AND FORMATION OF EMBRYOID BODIES ON A GELATIN-COATED DISH AND ON THE FEEDER LAYER IN BUFFALO (BUBALUS BUBALIS)

2006 ◽  
Vol 18 (2) ◽  
pp. 205 ◽  
Author(s):  
M. S. Chauhan ◽  
V. Verma ◽  
R. S. Manik ◽  
P. Palta ◽  
S. K. Singla ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bubalus Bubalis ◽  
Embryoid Bodies ◽  
Feeder Layer ◽  
Fetal Fibroblast ◽  
Group 2 ◽  
Group 1

Isolation and culture of embryo-derived cell lines have been reported in many mammals, however, there is not even a single report toward initiation of such work in buffalo (Bubalus bubalis). Therefore the present study was carried out to isolate the inner cell mass from in vitro-produced buffalo blastocysts and to grow this inner cell mass for formation of embryoid bodies in a gelatin-coated dish and on an homologous fetal fibroblast feeder layer. Immature buffalo oocytes were isolated from the slaughterhouse ovaries. In vitro production of blastocysts was carried as reported by Chauhan et al. (1999 J. Dairy Science 82, 918-926). A total of 26 buffalo blastocysts were produced in vitro. These blastocysts were transferred into a 100-mL drop of Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS) for further culturing. Thirteen blastocysts hatched on the next day of culture. The hatched mass was separated, suspended in Dulbecco's phosphate buffer saline (DPBS, without Ca++ and Mg++) containing 5% FBS + 0.25% trypsin and examined under the zoom stereomicroscope until disappearance of the trophectoderm cells. The remaining cells of seven blastocysts were cultured in DMEM supplemented with 20% FBS on 0.2% gelatin coated culture dish (group 1), and cells of six blastocysts in DMEM medium supplemented with 20% FBS were cultured on a mitomycin-c-treated (10 �L/mL) feeder layer (group 2), for 14 days. The isolated cells attached to the bottom of the dish in both the groups, spreading was noticed on Day 5 of the culture in group 1 and on Day 3 of culture in group 2. The attached cells were trypsinized using DPBS with 0.25% trypsin, isolated, and subcultured further. Attachment and spreading was noticed only in group 2 subcultured cells. The cellular integrity was homogeneous and the plasma membrane was clearly visible in group 2, but not in group 1. Less than 10% of the attached cells formed embryoid bodies in group 1, where as more than 30% attached cells in group 2 formed embryoid bodies; the latter expressed alkaline phosphatase activity and were blue after staining. These results indicate that the culturing of the inner cell mass on an homologous fetal fibroblast feeder layer is a better choice for production of embryonic stem cells in buffaloes.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts

Development ◽  
1978 ◽  
Vol 45 (1) ◽  
pp. 93-105
Author(s):  
Brigid Hogan ◽  
Rita Tilly
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Epithelial Layer ◽  
Visceral Endoderm ◽  
Limited Region ◽  
In Vitro Development ◽  
Vitro Development ◽  
Embryonic Ectoderm

This paper describes the in vitro development of inner cell masses isolated immunosurgically from mouse blastocysts which had been collected on 3·5 days p.c. and then incubated for 24 h. The inner cell masses continue to grow in culture and develop through a series of stages with increasing complexity of internal organization. By day 1 all of the cultured ICMs have an outer layer of endoderm, and by day 3 some of them have two distinct kinds of inside cells; a columnar epithelial layer and a thin hemisphere of elongated cells. Later, mesodermal cells appear to delaminate from a limited region of the columnar layer, close to where it forms a junction with the thinner cells. By day 5, about 25% of the cultured ICMs have a striking resemblance to normal 7·5-day p.c. C3H embryos, with embryonic ectoderm, extra-embryonic ectoderm and chorion, embryonic and extra-embryonic mesoderm, and visceral endoderm. When mechanically disrupted and grown as attached clumps of cells in a tissue dish, these embryo-like structures give rise to trophoblast-like giant cells. These results suggest that the inner cell mass of 4·5-day p.c. blastocysts contains cells which can give rise to trophoblast derivates in culture.

Leptin treatment of in vitro cultured embryos increases outgrowth rate of inner cell mass during embryonic stem cell derivation

2019 ◽  
Vol 55 (7) ◽  
pp. 473-481 ◽  
Author(s):  
Ali Cihan Taskin ◽  
Ahmet Kocabay ◽  
Ayyub Ebrahimi ◽  
Sercin Karahuseyinoglu ◽  
Gizem Nur Sahin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Stem Cell Derivation

Derivation of human embryonic stem cell lines after blastocyst microsurgery

2010 ◽  
Vol 88 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Guoliang Meng ◽  
Shiying Liu ◽  
Xiangyun Li ◽  
Roman Krawetz ◽  
Derrick E. Rancourt
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Clinical Medicine ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hesc Lines

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.

183 DEVELOPMENT OF PORCINE EMBRYOS MICROINJECTED WITH PORCINE EMBRYONIC GERM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 199
Author(s):  
C.-H. Park ◽  
S.-G. Lee ◽  
D.-H. Choi ◽  
M.-G. Kim ◽  
C. K. Lee
Keyword(s):  
Germ Cells ◽  
Fluorescent Protein ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Green Fluorescent Protein Gene ◽  
Cleavage Stage ◽  
Allocation Pattern

Embryonic germ (EG) cells, derived from primordial germ cells in the developing fetus, are similar to embryonic stem (ES) cells in terms of expression pattern of undifferentiated markers and their ability to colonize both the somatic and the germ cell lines following injection into a host blastocyst, which has been proven in mouse. Several studies using porcine EG cells have shown that it is possible to produce somatic chimeras after blastocyst injection. However, not only was the degree of reported chimerism low, but also there has been no report about the fate of injected EG cells in porcine blastocysts. This study was designed to observe the distribution pattern of porcine EG cells in chimeric blastocyst after injection into cleavage-stage porcine embryos. To ascertain development of microinjected porcine embryos with EG cells, 10 to 15 EG cells were injected into cleavage stage of in vitro fertilized embryos and cultured up to blastocyst. Also, porcine EG cells were labeled with DiO (Invitrogen, Carlsbad, CA) on the cell membrane or transfected with green fluorescent protein gene to observe whether the EG cells injected in the host embryo would incorporate into the inner cell mass (ICM) or trophectoderm (TE). Chimeric embryos were produced and allowed to develop into blastocysts to investigate the injected EG cells would come to lie in ICM and/or TE of the blastocyst, by scoring their position. In result, developmental rate was similar in all treatments. In all treatments, EG cells were mainly allocated in both ICM and TE of the chimeric blastocysts. These results suggest that examining the allocation pattern of injected EG cells, maintained pluripotency in vitro, could provide clues of differentiation process in vivo. Furthermore, to enhance the allocation of EG cells into the embryonic lineage, it would be required to optimize the culture condition for EG cells as well as embryos. Further experiment are needed to determine whether the injected EG cells could maintain their properties throughout the environment in the embryonic development in vitro. Table 1. Distribution of the porcine EG cells microinjected into cleavage-stage embryos

165 EXPRESSION OF Oct-4 IN BUFFALO (BUBALUS BUBALIS) EMBRYOS GENERATED THROUGH IN VITRO FERTILIZATION OR PARTHENOGENETIC ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 162
Author(s):  
D. Kumar ◽  
T. Anand ◽  
K. P. Singh ◽  
M. S. Chauhan ◽  
P. Palta ◽  
...  
Keyword(s):  
Relative Humidity ◽  
Inner Cell Mass ◽  
Polar Body ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bubalus Bubalis ◽  
Dnase I ◽  
Parthenogenetic Activation ◽  
Cell Lysate

Octamer-4 (Oct-4) is a member of Class V of the POU transcription factors family, which is involved in transcriptional regulation during early embryonic development and cell differentiation. It is expressed in the inner cell mass of blastocysts and in embryonic stem cells (ESCs), and its expression is widely used as a marker of pluripotency in ESCs in many species. This study was, therefore, carried out to examine the expression of Oct-4 in embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages generated through IVF or parthenogenetic activation. A total of 100 embryos were used in the study, 10 for each embryonic stage from both methods of embryo production. Immature oocytes obtained from slaughterhouse buffalo (Bubalus bubalis) ovaries were subjected to IVM in TCM-199 + 10% FBS + 5 µg mL–1 pFSH + 0.81 mm sodium pyruvate for 24 h in a CO2 incubator (5% O2, 5% CO2, 90–95% relative humidity) at 38.5�C. IVF was carried out immediately after IVM; the cleaved embryos were cultured for 8 days in CR2 medium containing 0.6% BSA and 10% FBS for production of embryos at different stages. For production of embryos through parthenogenesis, after 24 h of IVM, oocytes were denuded of cumulus cells by incubation in 0.2% hyaluronidase in Dulbecco's phosphate-buffered saline for 2 min. The denuded oocytes with a prominent polar body were parthenogenetically 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 in a CO2 incubator (5% O2, 5% CO2, 90–95% relative humidity) at 38.5�C, and then subjected to IVC as described above. A two-step RT-PCR was carried out using Cells-to-cDNA Kit-II (Ambion, Austin, TX, USA), using bovine primers 52-GTT CTC TTT GGA AAG GTG TTC-3' and 5'-ACA CTC GGA CCA CGT CTT TC-3' for the amplification of Oct-4. For this, the embryos were washed with PBS, transferred to 30 µL of cold cell lysis buffer and incubated in a thermal cycler at 75�C for 10 min. The cell lysate was treated with DNase-I at 37�C for 30 min to degrade genomic DNA and then heated at 75�C for 5 min to inactivate the DNase-I. The cell lysate (10 µL) was used for making cDNA using random primer. The PCR cycle included heating to 94�C for 2 min, followed by 33 cycles of 94�C for 30 s, 57�C for 30 s, and 72�C for 45 s. A final extension at 72�C for 10 min was carried out to complete the amplification of the Oct-4 gene. Transcripts of Oct-4 were detected at all of the embryonic stages, from the 2-cell through the hatched blastocyst stage in both IVF and parthenogenetically generated embryos. These results indicate that Oct-4, which is believed to be a reliable marker for pluripotency of ESCs in a number of species, is expressed in early cleavage-stage buffalo embryos and continues to be expressed in preimplantation-stage blastocysts.

396 Oct-4 EXPRESSION IN CAT INNER CELL MASS-DERIVED CELLS CULTURED IN MEDIUM WITH DIFFERENT PROTEIN SOURCES

2010 ◽  
Vol 22 (1) ◽  
pp. 354
Author(s):  
T. S. Rascado ◽  
J. F. Lima-Neto ◽  
S. E. R. S. Lorena ◽  
B. W. Minto ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Domestic Cat ◽  
Santa Cruz

The domestic cat can be used as a biological model for humans because of similarities in some disease and genetically transmitted conditions. Embryonic stem cells might complete nuclear reprogramming more efficiently than somatic cells and, therefore, are potentially useful for increasing interspecific cloning success. The objective of this study was to establish an effective culture system for inner cell mass (ICM)-derived cells in the domestic cat, testing the ability of the ICM to attach to the culture dish and to form embryonic stem cell colonies in the presence of fetal calf serum (FCS) and Knockout serum (KS). Moreover, knowing that the transcription factor Oct-4 is important for the maintenance of pluripotency in human and murine embryonic stem cells, the expression of this factor was evaluated in in vitro-produced blastocyst and in the attached ICM. Domestic cat oocytes were matured, fertilized, and cultured in vitro until the blastocyst stage. The ICM was mechanically isolated (n = 60) using a scalpel blade and transferred to a monolayer of chemically inactivated cat fibroblasts with 10 μg mL-1 mitomicin C. The base culture media (BM) was DMEM/F12 supplemented with nonessential amino acids, glutamine, leukemia inhibitory factor, fibroblast growth factor-2, 2-mercaptoethanol, and antibiotics. Three groups were tested: G1 = BM with 20% FCS (20); G2 = BM with 20% KS (20); G3 = BM with 15% FSC and 5% KS (20). Culture was performed in a 5% CO2 in air incubator at 38.5°C. No statistical difference was observed among groups in relation to ICM attachment (chi-square, P > 0.05). Ninety percent of the ICM presented good adhesion after 3 days of culture and started to grow in all media tested. However, until now, no good colonies were formed. Fifteen blastocysts and 10 attached ICM were fixed in 3% paraformaldehyde and permeabilized in 0.2% triton X-100 in PBS. Subsequently, to block nonspecific binding of the primary antibody, the preadsorption for 2 h at room temperature with OCT4 blocking peptide (sc-8628P, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. Samples were incubated with Oct4 antibody (N-19 : sc 8628, Santa Cruz Biotechnology) and with the appropriate secondary antibody (A21431, Invitrogen) and examined by fluorescence microscopy. Oct4 protein was detected both in the ICM and trophoderm cells, and it was distributed in cytoplasm and nuclei. These embryos were also stained with Hoechst 33342. Although further standardization of the culture media is needed, it seems that the KS can be replaced by FCS in cat embryonic stem cell culture. Furthermore, the immunostain of the trophoderm with Oct-4 indicates a difference in the expression of this factor when compared with its expression on human and murine blastocysts. This could be related to in vitro production, or Oct 4 is not a good pluripotency marker for cat embryos and cat embryonic stem cell, consequently. This fact has been noted in goat, bovine, and porcine embryos. Acknowledgment is given to FAPESP.

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