An autoradiographic analysis of the potency of embryonic ectoderm in the 8th day postimplantation mouse embryo

The potency of 8th day mouse embryonic ectoderm cells has been studied by injecting them into synchronous embryos which were subsequently cultured for 36 h. The development of injected embryos in vitro was comparable to that of embryos maintained in vivo. Tritiated thymidine was used to label the donor cells so that chimaerism could be analysed histologically. The results demonstrate the pluripotency of embryonic ectoderm in situ in the late primitive-streak-stage embryo. In addition, the patterns of donor cell colonization vary according to the site of origin and injection of the donor tissue.

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Carboxyfluorescein (CFSE) Labelling of Hepatocytes for Short-Term Localization following Intraportal Transplantation

Cell Transplantation ◽

10.1177/096368979400300506 ◽

1994 ◽

Vol 3 (5) ◽

pp. 397-408 ◽

Cited By ~ 32

Author(s):

Hikaru Fujioka ◽

Peter J. Hunt ◽

Jacek Rozga ◽

Guo-Du Wu ◽

Donald V. Cramer ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Isolated Hepatocytes ◽

Facs Analysis ◽

Short Term ◽

Tissue Sections ◽

Donor Cells ◽

Portal Vein Infusion

Renewed interest in the transplantation of isolated hepatocytes into the liver as a potential therapy for liver disease has stimulated the development of methods for the identification of donor cells within the recipient organ. We describe a method for cellular tagging and in vivo identification of intraportally transplanted hepatocytes using an intracellular fluorescent dye, 5(6)-carboxyfluorescein diacetate, succinimidyl-ester (CFSE). Rat and porcine hepatocytes were isolated and labelled with CFSE. The optimal conditions for labelling consisted of a buffered saline suspension of hepatocytes (5 × 106 cells/mL) in 20.0 μM CFSE incubated for 15 min at 37°C. In vitro, labelled hepatocytes were cultured either on fibronectin-coated chamber slides or in culture flasks. Cultures were evaluated in situ by fluorescence photomicrography or by fluorescence-activated cell sorting (FACS) after cell detachment. Cell viability was assessed serially and cultured, labelled hepatocytes retained the dye for up to 3 wk (last day of study). CFSE did not effect hepatocyte viability and there was no evidence of intercellular diffusion of the dye. In vivo, syngeneic Lewis rats underwent selective portal vein infusion of freshly isolated, labelled hepatocytes (2.0 × 107 cells/2.0 mL saline/animal) into the posterior liver lobes. All recipients were sacrificed 48 h and 96 h later and their livers examined. Transplanted hepatocytes were identified by fluorescence microscopy in tissue sections and by FACS following collagenase digestion of the liver tissue. CFSE persisted in a population of viable, engrafted hepatocytes. FACS analysis demonstrated that 9 ± 3% of the hepatocytes in the posterior liver lobes were labelled 48 and 96 h after transplantation. At 96 h following transplantation, multiple engrafted hepatocytes could be observed by fluorescence microscopy around the central veins. CFSE labelling allows for both in vitro identification and in vivo localization of donor hepatocytes. Furthermore, it appears to be more stable and specific for labelling hepatocytes than other tested dyes (especially DiI).

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Refrigeration of donor cells in preparation for bovine somatic nuclear transfer

Reproduction ◽

10.1530/rep.0.1220801 ◽

2001 ◽

pp. 801-808 ◽

Cited By ~ 5

Author(s):

JL Liu ◽

MK Wang ◽

QY Sun ◽

XR Zhang ◽

LK Jiang ◽

...

Keyword(s):

Nuclear Transfer ◽

Cultured Cells ◽

Donor Cell ◽

Serum Starvation ◽

Fresh Tissue ◽

Reconstructed Embryos ◽

Donor Cells ◽

Somatic Nuclear Transfer

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.

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16 EFFECT OF DONOR CELL AND TRICHOSTATIN A ON DEVELOPMENT OF CLONED DAIRY CATTLE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab16 ◽

2012 ◽

Vol 24 (1) ◽

pp. 119

Author(s):

Z. Mei-Ling ◽

Z. Yun-Hai ◽

T. Yong ◽

L. Ya ◽

C. Hong-Guo ◽

...

Keyword(s):

Trichostatin A ◽

Donor Cell ◽

Developmental Competence ◽

Donor Cells ◽

Significant Difference ◽

Blastocyst Rate ◽

Different Origins ◽

Vitro Development

The objective of present study was to investigate the effects of treatments to donor cells with fresh digestion (FD), cryopreservation/thawing (CT), trichostatin A (TSA) and durations of culture using TSA-CR1aa medium on in vitro development of dairy cow cloned embryos. In addition, some somatic cell cloned embryos were transferred to surrogates in heat to evaluate the in vivo developmental competence. The results (Table 1) showed that pretreatment of donor cells using TSA could significantly increase both cleavage and blastocyst rates of embryos (P < 0.05) compared with FD and CT group, whereas no significant difference was found between FD and CT group. When cloned embryos were subjected to TSA treatment in CR1aa for different times (0, 24, 48 and 60 h), the results showed that the blastocyst rate in the 60-h group was the highest (36.11 ± 1.78%) compared with the other groups (P < 0.05). Whereas the reconstructed embryos derived from donor cells treated with TSA for 24 h were continually cultured in TSA for different times (24, 48 and 60 h), the results showed that the blastocyst rate (37.39 ± 1.78%) in the 60-h group was significantly higher than that of the 24-h (25.48 ± 1.34%) group (P < 0.05). Finally, when the cloned embryos from different groups were respectively transferred to 40 natural oestrus recipients, no significant difference in terms of pregnancy rate among groups was found; however, a viable cloned calf was successfully obtained from TSA-treated donor cells and cloned embryo. Therefore, cloned embryos treated with optimized methods can develop to term. Table 1.Pregnancy results established from embryos of different origins

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24 EFFECT OF DONOR CELL TYPE ON IN VITRO AND IN VIVO DEVELOPMENTAL COMPETENCE OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab24 ◽

2016 ◽

Vol 28 (2) ◽

pp. 142

Author(s):

N. L. Selokar ◽

P. Sharma ◽

D. Kumar ◽

R. K. Sharma ◽

P. S. Yadav

Keyword(s):

Somatic Cell ◽

Seminal Plasma ◽

Donor Cell ◽

Cell Types ◽

Developmental Competence ◽

Cell Type ◽

Donor Cells ◽

Donor Cell Type

Selection of the donor cell type for somatic cell NT is very important based on its capability to be reprogrammed by the oocyte cytoplasm. A very wide variety of donor cells of different origin have been used for somatic cell NT, having differences in the overall efficiency. The aim of this study was to compare the cloning efficiency of donor cells derived from the ventral side of origin of tail skin and seminal plasma of a buffalo bull (age: 3 years old). Somatic cells from skin and seminal plasma were isolated and cultured as described by Selokar et al. (2014 PLOS ONE 9(3), e90755). Cultured seminal plasma cells had classic epithelial morphology, grew in clusters, were hexagonal in outline shape, and were positive for immunocytochemical detection of keratin marker, indicating that they were of epithelial origin, whereas tail-derived cells were spindle in shape and found positive for vimentin expression, indicating the fibroblast origin. To determine their reprogramming potential, these cells between passages 5 to 8 were used for the production of buffalo cloned embryos by handmade cloning as per the method described by Selokar et al. (2012 Theriogenology 78, 930–936). In brief, oocytes were isolated from slaughter-house ovaries and matured in vitro. After 21 h of maturation, cumulus cell mass and zona pellucida were removed by enzymatic treatment, hyaluronidase and pronase, respectively. Zona-free buffalo oocytes were enucleated on the basis of protrusion cone. A single somatic cell was attached to an enucleated oocyte with addition of phytohemagglutinin, followed by sandwich type of electrofusion between the somatic cell-bearing oocyte and enucleated oocyte using BTX electrofusion machine. Fused oocytes were activated by 4 μM calcium ionophore for 5 min and incubated in 2 mM 6-DMAP for 4 h and were cultured in K-RVCL-50® medium for 7 days on a flat surface in a 4-well dish in an incubator (5% CO2 and 38.5°C temperature). The total numbers of embryos reconstructed from tail-derived cells and semen-derived cells were 132 and 158, respectively. Cleavage and blastocyst rate were calculated from total embryos cultured, and data were analysed by Student’s t-test. We found no significant effect on both cleavage (89.30 ± 2.1 v. 94.1 ± 0.6) and blastocyst rate (40.7 ± 4.0 v. 43.1 ± 9.6) for the embryos produced from cells derived from tail and seminal plasma. To study the in vivo developmental competence of embryos derived from the 2 donor cell types, one embryo of each cell type was transferred into 6 recipient animals. Pregnancies were confirmed by ultrasonography at 30 to 35 days after transfer and monitored regularly at 15-day intervals up to 90 days. Three pregnancies were found for tail-derived cells, whereas no pregnancy was obtained for semen-derived cells. Out of 3 pregnancies obtained, 1 embryonic death was observed before 45 days, and 2 are continuing at advance stage. In conclusion, tail-derived cells are the better donor cell choice for buffalo somatic cell NT research. Currently, our focus is on epigenetic reprogramming behaviour of these 2 different cell types to elucidate the possible reprogramming mechanism.

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Ontogeny of hyaluronan secretion during early mouse development

Development ◽

10.1242/dev.117.2.483 ◽

1993 ◽

Vol 117 (2) ◽

pp. 483-492 ◽

Cited By ~ 1

Author(s):

J.J. Brown ◽

V.E. Papaioannou

Keyword(s):

Primitive Streak ◽

Synthetic Activity ◽

Visceral Endoderm ◽

Mouse Development ◽

Cell Lineages ◽

Early Mouse ◽

Serum Substitute ◽

Embryonic Ectoderm

The ontogeny of hyaluronan (HA) secretion during early mouse embryogenesis has been investigated using a biotin-labelled HA-binding complex from cartilage proteoglycan. HA is first secreted by visceral endoderm cells of the early egg cylinder on day 5.5 post coitum (p.c.), predominantly into the expanding yolk cavity. On day 6.5 p.c., HA is present in both the yolk and proamniotic cavities, but pericellular staining is restricted to the visceral endoderm and a population of embryonic ectoderm cells at the antimesometrial end of the proamniotic cavity. By the primitive streak stage, HA is secreted into the ectoplacental, exocoelomic, amniotic and yolk cavities, whilst the only cells exhibiting pericellular staining are those of the embryonic and extraembryonic mesoderm, including the allantois. Comparisons of HA-staining patterns of cultured whole blastocysts, microdissected trophectoderm fragments and immunosurgically isolated inner cell masses, revealed no trophoblast-associated HA secretion during outgrowth in vitro but significant synthetic activity by the endodermal derivatives of differentiating inner cell masses. To identify the cell lineages responsible for secretion of HA into the embryonic cavities and to investigate the origin of the HA observed around migrating mesoderm cells, day 7.5 p.c. primitive streak stage conceptuses were dissected into their various embryonic and extraembryonic cell lineages. HA secretion was observed after short-term suspension culture of mesoderm, embryonic ectoderm and embryonic endoderm, but was undetectable in fragments of ectoplacental cone, parietal yolk sac (primary giant trophoblast and parietal endoderm), extraembryonic ectoderm or extraembryonic endoderm. The level of synthesis by the HA-positive tissues was markedly enhanced by culture in medium containing serum, compared with that obtained following culture in medium supplemented with a defined serum substitute containing insulin, transferrin, selenous acid and linoleic acid. This suggests that additional growth factors, present in serum but absent from the serum substitute, are required for optimal HA synthesis by the HA-secreting tissues in vitro, and probably also in vivo. The implications of these events for implantation and the development of peri- and early post-implantation mouse embryos are discussed, and a new role for HA in the initial formation and expansion of the embryonic cavities is proposed.

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An autoradiographic analysis of tissue potency in different regions of the embryonic ectoderm during gastrulation in the mouse

Development ◽

10.1242/dev.69.1.265 ◽

1982 ◽

Vol 69 (1) ◽

pp. 265-285

Author(s):

R. S. P. Beddington

Keyword(s):

Posterior Part ◽

Heterotopic Transplantation ◽

Developmental Fate ◽

Donor Cells ◽

Autoradiographic Analysis ◽

Embryonic Ectoderm ◽

New Location

In vitro chimaeras have been produced by injecting [3H]thymidine-labelled 8th day embryonic ectoderm, derived from the anterior, distal or posterior regions of the egg cylinder,into unlabelled synchronous embryos. Injected embryos were cultured for 36 h and the distribution of donor cells was analysed autoradiographically. One series of orthotopic injections was carried out and the results indicate that the developmental fate of embryonic ectoderm in the posterior part of the embryo is to form mesoderm, both embryonic and extraembryonic. Heterotopic injections of distal and posterior embryonic ectoderm demonstrate that these tissues readily conform to the colonisation patterns characteristic of their new location. In contrast, anterior embryonic ectoderm showed some preference for definitive ectoderm differentiation following heterotopic transplantation. However, there was no evidence that the normal fate of tissue from the three regions studied could be explained by pre-existing mosaicism in the embryonic ectoderm.

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mRNA localization studies suggest that murine FGF-5 plays a role in gastrulation

Development ◽

10.1242/dev.112.2.407 ◽

1991 ◽

Vol 112 (2) ◽

pp. 407-415 ◽

Cited By ~ 13

Author(s):

J.M. Hebert ◽

M. Boyle ◽

G.R. Martin

Keyword(s):

Mouse Embryo ◽

Germ Layers ◽

Teratocarcinoma Cell ◽

Signalling Molecules ◽

Expression Studies ◽

Rna In Situ Hybridization ◽

Embryonic Ectoderm

During gastrulation in the mouse, the pluripotent embryonic ectoderm cells form the three primary germ layers, ectoderm, mesoderm and endoderm. Little is known about the mechanisms responsible for these processes, but evidence from previous studies in amphibians, as well as expression studies in mammals, suggest that signalling molecules of the Fibroblast Growth Factor (FGF) family may play a role in gastrulation. To determine whether this might be the case for FGF-5 in the mouse embryo, we carried out RNA in situ hybridization studies to determine when and where in the early postimplantation embryo the Fgf-5 gene is expressed. We chose to study this particular member of the FGF gene family because we had previously observed that its pattern of expression in cultures of teratocarcinoma cell aggregates is consistent with the proposal that Fgf-5 plays a role in gastrulation in vivo. The results reported here show that Fgf-5 expression increases dramatically in the pluripotent embryonic ectoderm just prior to gastrulation, is restricted to the cells forming the three primary germ layers during gastrulation, and is not detectable in any cells in the embryo once formation of the primary germ layers is virtually complete. Based on this provocative expression pattern and in light of what is known about the functions in vitro of other members of the FGF family, we hypothesize that in the mouse embryo Fgf-5 functions in an autocrine manner to stimulate the mobility of the cells that contribute to the embryonic germ layers or to render them competent to respond to other inductive or positional signals.

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In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083035 ◽

1978 ◽

Vol 36 (2) ◽

pp. 434-435

Author(s):

D. Reis ◽

B. Vian ◽

J. C. Roland

Keyword(s):

Cell Wall ◽

Self Assembly ◽

Plant Cell Wall ◽

Higher Plants ◽

Three Dimensional ◽

Cytochemical Study ◽

Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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