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Reproduction (2007) 133 207-218
DOI: 10.1530/REP-06-0013
Copyright © 2007 Society for Reproduction and Fertility
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RESEARCH

Foetal fibroblasts introduced to cleaving mouse embryos contribute to full-term development

Anna Piliszek, Jacek A Modlinski, Kazimiera Pysniak1 and Jolanta Karasiewicz

Department of Experimental Embryology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, 05-552 Wólka Kosowska, Poland and 1 Department of Genetics and Laboratory Animal Breeding, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Roentgen St, 5, Warsaw, Poland

Correspondence should be addressed to J Karasiewicz; Email: j.karasiewicz{at}ighz.pl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Foetal fibroblasts (FFs) labelled with vital fluorescent dye were microsurgically introduced into eight-cell mouse embryos, three cells to each embryo. FFs were first identified in the inner cell mass (ICM) in about one-third of embryos, whereas in three quarters of embryos FFs were located among trophoblast cells. Some elimination of FFs from trophoblast occurred later on. Eventually, in blastocysts’ outgrowths, an equally high contribution from FFs progeny (60%) was found in both ICM and trophoblast. Three days after manipulation, FFs resumed proliferation in vitro. More than three FFs were found in 46.2% of embryos on day 4. On the 7th day in vitro in 70% of embryos more than 12 FFs were found, proving at least three cell divisions.

To study postimplantation development, the embryos with FFs were transferred to pseudopregnant recipients a day after manipulation. After implantation, FFs were identified by electrophoresis for isozymes of glucose phosphate isomerase (GPI). A single 11-day embryo delayed to day 8 proved chimeric by expressing both donor isozyme GPI-1B and recipient GPI-1A. Similar chimerism was found in the extraembryonic lineage of 11% of embryos by day 12. Starting from day 11 onwards, in 32% of normal embryos and in 57% of foetal membranes, hybrid GPI-1AB isozyme, as well as recipient isozyme, was present. Hybrid GPI-1AB can only be produced in hybrid cells derived by cell fusion, therefore, we suggest that during postimplantation development, FFs are rescued by fusion with recipient cells. In the mice born, hybrid isozyme was found in several tissues, including brain, lung, gut and kidney.

We conclude that somatic cells (FFs) can proliferate in earlyembryonic environment until early postimplantation stages. Foetuses and the mice born are chimeras between recipient cells and hybrid cells with contributions from the donor FFs. Transdifferentiation as opposed to reprogramming by cell fusion can be considered as underlying cellular processes in these chimeras.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Totipotency of cells is their ability to differentiate into any cell type. Mouse embryonic stem (ES) cells (Evans & Kaufman 1981, Martin 1981) have been proven totipotent by obtaining mice developed entirely from these cells (Nagy et al. 1990, 1993, Modlinski et al. 1996). When injected into mouse blastocyst, a single stem cell of teratocarcinoma populated a variety of different tissues in adult mouse (Illmensee & Mintz 1976), showing its pluripotency.

Tissue stem cells have also revealed some potential to participate in the formation of various tissues when injected into blastocysts (‘blastocyst complementation test’). The first to conduct such a test was Brinster (1974), who injected bone marrow cells into mouse blastocysts finding a delay in transplant rejection from the donor strain in potentially chimeric mice born. After injecting adult haematopoietic stem cells (HSCs) into blastocysts, chimeric animals carried the progeny of HSCs in various haematopoietic tissues and their HSCs from bone marrow expressed embryonic and foetal globins (Geiger et al. 1998). After injection of HSCs from human cord blood cells into murine blastocysts, human donor contribution persisted to adulthood (Harder et al. 2002). Neurospheres obtained by aggregation of adult neural stem cells and introduced into blastocysts contributed to all three germ layers in 11-day-old mouse foetuses showing tissue-specific protein expression (Clarke et al. 2000). Multipotent adult progenitor cells (MAPCs) were found in many tissues of adult mice as a result of blastocyst complementation test (Jiang et al. 2002). Indeed, MAPCs could give rise to almost all types of cells, which was confirmed by transplanting single MAPC into blastocysts. Kues et al.(2005) have described cells that are derived from foetuses under conditions of overgrowth and oversupply of foetal calf serum in the culture medium as ‘foetal somatic stem cells (FSSCs)’. After injection into mouse blastocysts, such cells participated in tissues of the mesodermal lineage in 15-day embryos.

Studies on the potencies of non-stem somatic cells have been limited so far. Non-stem somatic cells can supply nuclei for cloning by nuclear transfer (Wilmut et al. 1997, Wakayama et al. 1998), which brings evidence in favour of their totipotency. However, it remains to be elucidated, if entire somatic cells can transdifferentiate to become totipotent and to participate in embryonic development. Hakelien et al.(2002) have shown that cytoplasmic extracts from one type of somatic cells (T lymphocytes, neuronal precursors) can reprogram other somatic cells (fibroblasts) in vitro. Fibroblasts treated with such cellular extracts show expression of transcription factors and antigens typical of donor cell extract. But can the environment of early embryos act similarly, or in other words, can reprogramming occur in vivo? Soon after somatic (epithelial) cells were injected into cleaving mouse embryos, gap junctions were found between them and the blastomeres of chimeric morulae and the injected cells were expressing totipotency marker Oct-4 (Burnside & Collas 2002). Interestingly, this early response was absent from fibroblasts after they had been injected. In this paper, we follow fibroblasts injected into cleaving mouse embryos to determine their long-term response to embryonic environment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Obtaining embryos
All animals originated from the mouse-raising facility in the Department of Experimental Embryology. Mice were kept under a 12-h day starting at 0700 h. Experiments were performed with the permission of the Third Local Ethical Committee in Warsaw. Inbred DBA/2 or MIZ (Swiss albino) females aged 2–3 months mated to 3- to 10-month-old males of the same breeds served as donors of embryos. To obtain eight-cell embryos, females were caged with males in the evening and next morning they were inspected for vaginal plugs. Those females that mated were killed by cervical dislocation 48–50 h later. Oviducts were excised into M2 manipulation medium (HEPES-buffered M16; Fulton & Whittingham 1978) and their contents were flushed with the same medium using a pipette introduced into the infundibulum.

Obtaining foetal fibroblasts
To establish primary cultures of somatic cells (foetal fibroblasts, FFs), females of C57BL10 or CBA/H strains were mated with males of the same or the other strain to obtain either C57BL10 foetuses or F1 (C57BL10xCBA/H) or F1 (CBA/HxC57BL10) foetuses. At day 13 or 14, p.c. females were killed by cervical dislocation and uterine horns were cut out and placed in sterile PBS. After the foetuses were dissected out of their foetal membranes, they were decapitated and eviscerated. The carcasses were rinsed in PBS, cut into small pieces and transferred to 0.25% trypsin/EDTA for 30 min at 37 °C. Trypsin digestion was stopped by adding Dulbecco’s modified Eagle medium (DMEM, Sigma)+10% foetal calf serum (FCS, Gibco). After centrifugation for 10 min at 200 g, the cells were suspended in the same medium and seeded onto tissue culture Petri dishes (cell suspension from single carcass per one 6 cm dish) to be cultured at 37 °C, in an atmosphere of 5% CO2 in air. Upon reaching confluence, the cells were passaged and were frozen at passages 3–6 to be used later. For the experiments, an ampoule of frozen cells was thawed and the seeded cells were cultured until reaching confluence. For manipulation, confluent culture was trypsinised, centrifuged (in some experiments the cells were stained with PKH26-GL at this stage) and suspended in a small amount (200 µl) of M2 medium to be taken to the manipulation chamber.

Membrane lipophilic dye PKH26-GL
PKH26-GL (Horan & Slezak 1989; Sigma) is a vital fluorescent lipophilic dye, persisting in cell membranes as long as 100 days after staining. Fibroblasts were stained with this dye before being introduced into the embryos, according to manufacturer’s instructions. Embryos with stained cells were observed under inverted Opto IM35 microscope (Zeiss) using phase-contrast optics and u.v. light (excitation filter 546 nm).

Manipulation
Before manipulation, the embryos were incubated (30 min–1 h) in M2 containing cytochalasin D (CD, Sigma, 1 µg/ml), at 37 °C in an atmosphere of 5% CO2 in air. Embryos together with 2 µl fibroblast suspension were placed in a drop of M2 medium with CD under paraffin oil in the manipulation chamber. Manipulation was performed at room temperature, as described by Stewart (1993), under Fluovert (Leitz, Wetzlar, Germany) inverted microscope, with differential interference Nomarski contrast (DIC). Mechanical Leitz (Germany) manipulator, micropumps: Beaudouin (France), connected with injection pipette, and CellTram Air, Eppendorf (Hamburg, Germany), connected with holding pipette, were used. Both holding and injection pipettes were prepared from thin-walled borosilicate capillaries of external diameter 1 mm, made of silica glass (GC 100T-15, Harvard Apparatus Ltd; Edenbridge, Kent, UK). Three fibroblasts were injected into the centre of each eight-cell embryo.

Culture conditions
After thorough rinsing with M2 without CD, the embryos were placed in drops of M2 or KSOM medium (Specialty Media, Phillipsburg, NJ, USA) under paraffin oil (Sigma) in Petri dishes (Corning) and were cultured at 37 °C, in an atmosphere of 5% CO2 in air. For outgrowth formation, blastocysts were placed in wells of 4- or 24-well dishes (Nunc or Corning) on feeder layer and were cultured in DMEM with high glucose (4.5 g/l), 0.1 mM ß-mercaptoethanol (Sigma), 0.1 mM non-essential amino acids (Sigma) and nucleotides (Sigma; Robertson 1987). Media supplements included 20% foetal calf serum (Gibco) and 1000 i.u./ml mouse leukaemia inhibitory factor (LIF; Sigma). For feeder layer production, confluent FFs were inactivated with 10 µg/ml mitomycin C (Sigma) for 2.5–3 h and were used either directly (5x104 cells/cm2) or after freezing.

In vitro studies
MIZ embryos containing F1 (C57BL10xCBA/H) or F1 (CBA/HxC57BL10) fibroblasts stained with PKH26-GL were cultured in vitro and were observed every 24 h to localise introduced cells. On day 4 of culture, the embryos were transferred onto feeder layers enabling their settlement and outgrowths formation, and were observed until day 10 (day of fibroblast injection, day 0). To limit deleterious effects of u.v. light, each embryo was observed not more than thrice, usually twice throughout its whole culture period.

Embryo transfer
For embryo transfer, F1 (C57BL10xCBA/H) or F1 (CBA/HxC57BL10) females mated to vasectomised F1 males were used as recipients. The embryos (5–12 per transfer) were transferred to the oviducts of recipients during 1st day of pseudopregnancy. Females were anaesthetised by i.p. injections of 0.1 ml/g body weight of 12.5% solution of Vetbutal (Biovet, Pulawy, Poland) in PBS.

In vivo studies
After 24 h of culture, DBA/2 embryos with C57BL10 fibroblasts were transferred to pseudopregnant females and autopsy was performed at days 8–14. Morphological observations were followed by collecting material for electrophoresis of GPI isozymes. Few recipients were left to bear the young. Animals born, after they grew up, were autopsied and tissue samples were frozen for electrophoresis. After 24 h of culture, MIZ embryos with F1 (C57BL10xCBA/H) or F1 (CBA/HxC57BL10) fibroblasts inside were transferred to recipients which were left undisturbed until birth of young. Young born, after reaching puberty, were crossed with mates from the strain of donors of embryos.

Morphological evaluation of development and collecting tissue samples for GPI analysis
Embryos and foetuses were observed under stereomicroscope in both transmission- and epi-illumination to evaluate their developmental stage (according to Kaufman 1992). After morphological evaluation, 8-day egg cylinders were cleared of Reichert’s membrane (which was frozen as a whole) and cut with a glass needle into embryonic and extraembryonic halves to be frozen separately. Samples of amnion, yolk sac and/or the embryos to be frozen separately were taken from 11-to 14-day implantation sites. In case of resorption of the embryo, tissue remnants were taken as samples. From animals born, some organs were dissected out and tissue fragments of about 1 mm diameter were stored.

Electrophoresis of GPI
The isozymes of GPI (DeLorenzo & Ruddle 1969, Lyon & Searle 1989) served as markers of donor and recipient cells in presumed chimeras. Electrophoresis of GPI was performed according to the method by Buehr & McLaren (1985) in samples of DBA/2 strain (GPI-1A) embryos injected with C57BL10 fibroblasts (GPI-1B). Tissue samples were first rinsed in PBS and then in Tris–glycine buffer (0.025 M) to remove any traces of blood. They were placed in 0.5 ml Eppendorf tubes containing 10–200 µl buffer and were frozen at –20 °C. Samples of blood of animals born taken from tail tips were frozen and thawed thrice in distilled water (1:1 volumes) before being stored at –70 °C. For placing on electrophoretic plate, the samples were thawed and frozen thrice in Tris–glycine buffer (0.025 M, Sigma) and 0.5 µl liquefied sample was applied. Blood of DBA/2 (GPI-1A) and C57BL10 (GPI-1B) animals served as reference samples. Cellulose acetate-covered plates (Titan III H, Helena Biosciences, Gates-head, Great Britain) were soaked in low molar Tris–glycine buffer (0.025 M) for 20 min at room temperature. Electrophoresis was performed in Tris–glycine buffer (0.1 M) for 60 min, at room temperature, at 200 V in ZipZone chamber (Helena Laboratories, Beaumont, TX, USA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
In vitro development
FFs originally placed in the centre of each embryo were found in either ICM or trophoblast or both of them after 24 h. The percentage of embryos with fibroblasts located within an outer layer was almost twice as high when compared with fibroblasts in the centre of embryos. This ratio did not change throughout 3 days of culture (Table 1Go), suggesting no migration of introduced cells within embryos. The percentage of embryos with FFs in ICM remained constant from days 1 to 5. However, between days 2 and 5 there was a decline in the percentage of embryos with fluorescent cells in trophoblast, indicating elimination of introduced cells and their progeny from trophoblast. Therefore, starting from day 4 onwards, the fraction of embryos with FFs in ICM was comparable with those containing cells in trophoblast (Table 1Go). At days 6–10, the percentages of embryos with introduced cells either in ICM or in trophoblast had increased (Table 1Go). This might be the result of enhancing effects of feeder layer and LIF medium on cell division upon outgrowth formation at day 6.


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  		Table 1 Contribution of fibroblasts (FFs) and/or their daughter cells to inner cell mass (ICM) and trophoblast.
 
Contribution of FFs and/or their daughter cells to embryos was expressed as the percentage of embryos containing at least one fluorescent cell at subsequent days of culture. The day after introducing three FFs, a decline below this number was found in about 30% of embryos (Table 2Go, day 1), indicating some deterioration of introduced fibroblasts. Only in 6 and 17% of embryos, more than three fluorescent cells were found at days 1 and 2 respectively. This suggests an inhibition of FFs’ divisions concurrent with cleavage delay in recipient embryos. At the fourth day, almost half of the embryos (46.2%) harboured more than three fibroblasts and this percentage increased every day reaching 100% at day 7. A slight decrease occurred later, which might suggest deleterious influence of prolonged culture. Presumed numbers of cell divisions undergone by FFs in cultured embryos are shown in Fig. 1Go and Table 2Go. Embryos with six or more fluorescent cells appear at day 2 indicating two cell divisions and on day 3, embryos with 12 cells indicating three divisions appear. After 7 days, in 70% of embryos fibroblasts underwent at least three divisions.


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  		Table 2 Contribution of fibroblasts (FFs) and/or their daughter cells to embryos cultured in vitro.
 

Figure 1
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  		Figure 1 Diagram showing presumed number of cell divisions undergone by injected fibroblasts (FFs) in mouse embryos cultured in vitro. Three fibroblasts were injected to eight-cell embryos and on subsequent days of culture (x-axis), fractions of embryos with PKH26-marked cells were counted. The assumption was that the presence of 4–6 marked cells (yellow) indicates one division, of 7–12 cells (orange) indicates two divisions and the presence of more than 12 cells (brown) indicates three divisions underwent by injected cells. y-Axis shows the percentage of embryos containing marked cells, for example, on day 3, 10.6% of embryos contained 4–6 cells (underwent one division), 0.9% of embryos harboured 7–12 cells (underwent two divisions) and 5.3% of embryos harboured more than 12 cells (underwent three divisions).

 
A total of 820 embryos were injected with FFs. Over 99% embryos survived until next day. However, they were only at the 8- to 16-cell stages, and they reached the blastocyst stage not earlier than after the following 24 h. Hatching in vitro was impaired in some blastocysts, probably due to perforation of the zona pellucida during manipulation. After placing them onto the feeder layer, the embryos attached during an overnight culture and formed outgrowths after next day. Fibroblasts stained uniformly with PKH26-GL and this pattern persisted 3 days in vitro (Fig. 2Go). Later on, fluorescent patches appeared (Fig. 3Go). Patchy distribution, although normal in accordance with the manufacturer’s information (Sigma), interfered in some cases with counting fluorescent cells within outgrowths, thus reducing the numbers of outgrowths analysed at days 5–10.


Figure 2
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  		Figure 2 Foetal fibroblasts (FFs) in mouse blastocysts. (A) Hatching blastocyst at the third day after FFs injection (phase contrast). (B) The same image after merging with u.v.-light picture; red fluorescent FFs are present in the inner cell mass (left) and in trophoblast (up and middle). (C) Hatched blastocyst at the fourth day after FFs injection. (D) The same image after merging with u.v.-light view; FFs can be seen in inner cell mass (left) and in trophoblast (right).

 

Figure 3
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  		Figure 3 Foetal fibroblasts (FFs) in mouse blastocysts’ outgrowths at days 6 and 7 after injection. Phase-contrast images on the left (A, C and E) and the same images after merging with u.v.-light pictures – on the right (B, D and F). Note a flattened, fluorescent, trophoblast-like cell in F. Patchy distribution of the stain is seen in this cell.

 
In vivo development
In 19 transfers, 172 DBA/2 embryos were transplanted, giving rise to 101 implantation sites (58.7%). Out of 72 implantation sites taken for GPI analysis, in 62 the embryos were found (see Table 3Go).


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  		Table 3 Postimplantation development of FFs-injected embryos subjected to GPI analysis.
 
Chimerism was confirmed by co-occurrence of recipient isozyme GPI-1A with donor isozyme GPI-1B in one embryo recovered at day 11, which was severely delayed to the stage of 8-day egg cylinder and showed signs of degeneration (Table 4Go). Otherwise, co-occurrence of both isozymes was only confirmed in extraembryonic lineage. These comprised: twice – in Reichert’s membrane (of 8-day egg cylinders: one normal and one delayed) and five times – in the amnion (once of an 11-day normal embryo and four times of 12-day normal or delayed or abnormal foetuses). The last two cases of confirmed chimerism were in tissue remnants after resorption.


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  		Table 4 Contribution of GPI isozymes to embryos/foetuses and foetal membranesa.
 
Surprisingly, in four implantation sites, only the donor isoform GPI-1B was found. Three of them comprised remnants after resorption, found at days 8–12. In one case, some tissue found on day 11 appeared not dissimilar to an egg cylinder (Table 4Go).

In the samples taken from 11- to 14-day implantation sites, an isozyme was found on electrophoretic plates that migrated intermediately between the slow and the fast variants. Based on its speed of migration, the isozyme was recognised as hybrid GPI-1AB. Hybrid GPI-1AB isozyme was found in morphologically normal embryos (Fig. 4A–CGo). It was also found in one delayed 12-day embryo, whose amnion contained both GPI-1A and GPI-1B, without GPI-1AB contribution. Among organ samples, GPI-1AB was found in the heart, head and the liver of foetuses. Hybrid isozyme was also detected in the amnion of 12-day (Fig. 4DGo) and 13-day normal foetuses (six cases) and in the amnion of a 13-day abnormal foetus whose yolk sac was chimeric (one case). GPI-1AB appeared frequently in the yolk sac of 11- to 14-day foetuses (69.3% of 49 samples). Twelve-day embryos showing GPI-1AB in their foetal membranes were morphologically normal in almost 80% of cases. However, among 13-day embryos that contained GPI-1AB in their yolk sac (a total of eight embryos), only 50% were considered morphologically normal. On day 14, GPI-1AB was also found in yolk sacs of resorbed embryos.


Figure 4
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  		Figure 4 Postimplantation development of FFs-injected embryos and electrophoretic analysis. (A and B) Normal day 12 chimeric mouse foetuses. (C) Electrophoretic plate showing heterodimer GPI-1AB in such foetuses; 1 and 5, GPI standard: mixed blood samples from DBA/2 and C57BL10 animals; 2, tissue sample from foetus A; 3, tissue sample from foetus B; 4, tissue sample from yet another normal 12-day foetus. (D) Electrophoretic plate showing heterodimer GPI-1AB in foetal membranes of 12-day normal foetuses developed from FFs-injected embryos; these foetuses themselves were not subjected to GPI analysis; 1 and 7, GPI standard: mixed blood samples from DBA/2 and C57BL10 animals; 2, tissue sample from amnion; 3, tissue sample from yolk sac; 4, tissue sample from another amnion; 5, tissue sample from another yolk sac; 6, tissue sample from yet another yolk sac. (E) Electrophoretic plate showing heterodimer GPI-1AB in brain tissue of animals born after FFs injection into mouse embryos; 1 and 5, GPI standard: mixed blood samples from DBA/2 and C57BL10 animals; 2, sample from brain of a fertile male (animal 3 in Table 5Go); 3, brain of an infertile female (animal 4 in Table 4Go); 4, brain of a fertile female (animal 5 in Table 5Go). GPI-1A, recipient isozyme; GPI-1B, donor isozyme; GPI-1AB, hybrid isozyme. (F) A tumour found in chimeric DBA/2 female (animal 5 in Table 5Go); lateral (left) and frontal (right) view.

 
Animals reaching full-term
In 43 transfers, 325 embryos were transplanted giving rise to 70 young born (21.5%). Eleven of them were eaten by mothers soon after birth. Others were allowed to develop. One of the young was killed at 1 month due to developmental retardation and poor health. Males comprised 75% of young. Four females and one male were considered sterile. Fourteen animals (ten females and four males) died at 3–20 months of age for unknown reasons.

Donor FF-derived pigmentation was not found in the skin of adult chimeras. Progeny of fertile animals have not revealed such pigmentation either. Electrophoretic analysis of GPI was performed in tissue samples from five DBA/2 presumed chimeras (Table 5Go). All these animals revealed hybrid GPI-1AB and recipient GPI-1A isozyme. GPI-1AB was present in the organs originating from all three germ layers: mesoderm – kidneys, heart, gonads and blood; mesoderm/ectoderm – skin; mesoderm/ endoderm – liver, endoderm – lung and intestine and ectoderm – brain (Fig. 4EGo). In some cases, donor GPI-1B isozyme co-occurred with hybrid and recipient isoforms. In the skin of the newborn and of the 1-month-old chimera hybrid isozyme was found. It was also found in the gonads of all animals except the newborn one. GPI-1AB and GPI-1B were found in the blood of three animals, but two remaining ones showed only recipient isozyme GPI-1A in their blood.


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  		Table 5 GPI isozymes in tissues/organs of mice born.
 
Five females (out of 70 mice born after transfer of manipulated embryos) developed tumours. Four of them (two MIZ and two DBA/2 females) were killed and their internal organs were examined. In all the examined animals, the liver and the spleen were markedly enlarged. One of MIZ females at 10 months of age carried five tumours, four of which were found in four groins. Histopathological examination revealed lymphoblastomas. Liver and kidney were infiltrated with lymphocytes and in the ovary dysplasia of theca and follicular cells was found. The second MIZ female at 6 months of age had a tumour in right proximal groin, which was also diagnosed as lymphoblastoma. The liver was severely infiltrated by lymphocytes. In a sterile DBA/2 female (animal 4, Table 5Go) at the age of 16 months, a tumour was found of a diameter of about 2 cm, located extraperitoneally in the right hind groin under the skin. It was diagnosed as adenocarcinoma type B originating from mammary gland. Electrophoretic analysis confirmed the presence of hybrid isozyme GPI-1AB in the outer, dermal part of the tumour and in other tissues of this animal. Severely enlarged spleen (to 2.5 cm) as well as ovarian cysts (one of them sized 1.5x 1 cm, filled with blood on the left ovary; the second one 0.7x1 cm, filled with translucent fluid on the right) were found. The second DBA/2 female (animal 5, Table 5Go) at the age of 17 months had a large spherical tumour (Fig. 4FGo) located subcutaneously extraperitoneally near lymph nodes of left hind leg. This tumour was more solid than the remaining ones. Electrophoretic analysis revealed the presence of hybrid GPI-1AB in the solid part of the tumour as well as in other tissues of this animal. Enlarged spleen as well as right ovary cyst (filled with translucent fluid) was found accompanied by enlargement of kidneys.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Proliferation of fibroblasts in preimplantation embryos
The percentage of embryos harbouring more cells than the introduced three reached 40% only after 4 days of culture. The cell cycle of FFs at low passages takes about 20 h (Hernandez et al. 2003), the duration which is sufficient for fibroblasts to divide at least thrice throughout this period. Therefore, we conclude that inhibition of cell division occurs in deposited FFs, possibly due to signals from blastomeres. Burnside & Collas (2002) observed similar block of division of both fibroblasts and epithelial cells 24 h after introduction to cleaving embryos. On the other hand, proliferation of ES cells in blastocysts appears to depend on their number, when a single cell is injected, then it divides within 24 h, whereas two to five cells injected, do not (Saburi et al. 1997).

However, after FFs are introduced into embryos, proliferation in the latter is also inhibited. When cleaving embryos obtain ES cells, after 24 h they reach the blastocyst stage (Tokunaga & Tsunoda 1992, Stewart 1993, Saburi et al. 1997). This is not the case after somatic cells are injected (Burnside & Collas 2002; and our results). There is experimental evidence for asynchronous blastomeres affecting themselves in chimeras, when 1/2 and 1/8 blastomeres are aggregated, cavitation occurs at intermediate time expected for either embryonic partner (Prather & First 1986). The delay in blastomere proliferation in our experiment might be the effect of prolonged blastomere cell cycle, due to the influence of the longer FFs’ cell cycle (fibroblasts in vitro – 20 h: Hernandez et al. 2003, blastomeres from 8- and 16-cell stages blastomeres – about 13 h: Lehtonen 1980, MacQueen & Johnson 1983). At the end of in vitro culture, up to 15 fluorescent cells were present in our chimeric embryos. This is about 3% of cells present in normal 8-day embryos in vivo (500 cells; Snow 1978).

Donor–recipient chimerism in egg cylinders and foetal membranes
The only case of chimerism between recipient (GPI-1A) and donor (GPI-1B) cells in the embryonic part was an egg cylinder, delayed 3 days in development and showing signs of degeneration. Otherwise, this type of chimerism was only found in Reichert’s membrane and later on, in the amnion at day 12. Thus, it seems that the presence of foreign cells is deleterious for the embryo proper but it does not impair embryonic development when present in the amnion until day 12. Contrary to somatic cells (FFs), when embryo-derived totipotent ES cells were injected into blastocysts, postimplantation chimerism was high not only in extraembryonic mesoderm, but also in embryonic body, germ line included (Beddington & Robertson 1989).

Rescue of fibroblasts by fusion with recipient cells?
Starting from the 11th day of development on, both the embryos/foetuses (32%) and their foetal membranes (57%) revealed the presence of hybrid isozyme GPI-1AB. Moreover, the mice born and grown-up revealed hybrid isozyme in their tissues. This can only occur when in one cell both variants (GPI-1A and GPI-1B) are produced to form heteropolymer GPI-1AB, i.e. in heterozygotes or in hybrid cells (Lyon & Searle 1989). Animals used in our experiments were not heterozygous for GPI, and therefore, indication for hybrid cells arises. The presence of the heterodimeric type of GPI was earlier treated as evidence of cell fusion between trophoblast cells (Gearhart & Mintz 1972a) and between myoblasts upon eye muscle formation (Gearhart & Mintz 1972b) in mouse development. We suggest that also in our experimental embryos, hybrid cells arise due to cell fusion. We further hypothesise, that starting from day 11 on, the experimental foetuses and mice are chimeras between recipient cells and hybrid cells derived from fusion offibroblasts with recipient cells. In experimentally constructed tetraploid embryos, hybrid GPI-1AB isozyme could only be detected 3 days after cell fusion, since earlier in embryogenesis only the isozymes of both partners were present (Petzoldt 1991). This observation suggests that in our experiment, cell fusion could occur earlier than at day 11, when hybrid isozyme was first demonstrated. Indeed, if we accept that chimeric GPI-1A{leftrightarrow}GPI-1B embryos only survive until days 7–8, then this is the last moment for fibroblast progeny to be rescued by fusion with recipient cells. The absence of hybrid GPI-1AB isozyme from days 7 to 8 egg cylinders found in this study may be due to delayed expression (like that described above) or to insufficient number of hybrid cells, falling below 1% of the studied sample, which precludes detection in GPI electrophoretic identification (Chapman et al. 1972). GPI isozymes were early (Chapman et al. 1972, Gearhart & Mintz 1972a, 1972b) and continuously (West & Flockhart 1994, Wang et al. 1997, Suwinska et al. 2005) used as markers of chimerism in the mouse. In day 9, embryonic chimeras that developed after aggregation of eight-cell embryos varying in GPI isozymes, no heterodimeric form was found thus excluding cell fusion until this stage (Gearhart & Mintz 1972a). Balinsky et al.(1983) describe GPI isozyme patterns in chimeric (‘allophenic’) mice and show that the hybrid isozyme appears in a tissue (lymphocytes) of some (two) of these mice. It is possibly the same phenomenon as we noticed in our study.

It remains to be studied, why mice born in our experiments, which were 75% males and were proven chimeric, did not reveal coat colour chimerism. Over-representation of males in the progeny of mice is a strong indication towards chimerism (Tarkowski 1961), since mice chimeric in chromosomal sex (XX{leftrightarrow}XY) are not hermaphrodites, but they are fertile males instead. Cells of XY constitution in the gonad determine its differentiation into testis (for review, see McLaren (1984)). Triploid cells in chimeras express coat colour markers (Suwinska et al. 2005). Perhaps, hybrid tetraploid cells derived from FFs do not enter the skin or they do not produce pigment there.

Hybrid GPI-1AB isozyme was found in the gonads of chimeric animals, including one fertile male. However, neither GPI analysis of the progeny of this male was done nor can one expect that tetraploid cells can properly undergo meiosis to form functional gametes.

Reprogramming of hybrid somatic cells in the embryos
In our study, hybrid GPI-1AB isozyme was found in tissues originating not only from mesoderm, which is the layer of origin of fibroblasts, but also from two other germ layers; in a 13-day foetus, the heteropolymer was present in partially endodermal liver, and in born animals – in endodermal lungs, ectodermal brain and also in liver. However, in chimeric 10.5- to 15.5-day foetuses obtained after transfer of ‘foetal somatic stem cells (FSSCs)’ to blastocysts (Kues et al. 2005) only in muscle, genital ridge and liver contribution of introduced cells was found, suggesting their common mesodermal roots. ‘Foetal somatic stem cells (FSSCs)’ were derived in vitro from mouse foetuses under conditions of overgrowth and oversupply of foetal calf serum in the culture medium. In our study, FFs were derived from foetuses under standard tissue culture conditions (Freshney 1987). Presumed stem cells comprise only 1% of human FFs grown in vitro in standard conditions (Rieske et al. 2005). Therefore, it is most likely that 99% of mouse FFs used in our study are somatic cells.

Experimentally produced hybrid mouse cells, obtained in vitro by fusion of somatic cells (adult thymocytes) with ESCs and subjected to blastocyst complementation essay, survived in 7.5-day foetuses contributing to tissues originating from all germ layers (Tada et al. 2001). In addition, hybrids between foetal neural stem cells and ESCs in blastocyst complementation essay produced overt chimeras and the descendants of injected cells were detected in gut, kidney, heart and liver (where tetraploid cells are probably not eliminated; Ying et al. 2002). This comparison shows that FFs are reprogrammed as effectively in mouse embryos as are hybrids with contribution of totipotent ES cells.

Hybrids between ESCs and somatic cells are tetraploid (Terada et al. 2002), the condition presumably shared with hybrids between embryonic cells and FFs. Experimental tetraploid embryos develop until mid-pregnancy (Snow 1975, Tarkowski et al. 1977, Kaufman & Webb 1990) and in diplo{leftrightarrow}tetraploid chimeras 4N cells are gradually eliminated from embryonic body and remain only in extraembryonic structures (Tarkowski et al. 1977, Nagy et al. 1993). However, participation of tetraploid cells in normal mouse development has been later proven to depend on the way of producing chimeras and/ or on the genetic background of the donor. In embryonic chimeras formed by aggregation of two eight-cell diploid embryos with one four-cell tetraploid ones (Goto et al. 2002, Suemori et al. 1990), they were present in heart, liver, skin and intestinal epithelium of newborns. After embryonic stem cells were aggregated with tetraploid carrier blastomeres, one line, namely D3, promoted survival of tetraploid component in liver, lung and heart (Wang et al. 1997). The presence of tetraploid cells in epiblast, earlier considered impossible, has also been confirmed (Eakin et al. 2005). These data indicate that tetraploid cells are compatible with mouse development until birth.

Transdifferentiation or reprogramming by cell fusion?
Dedifferentiation of tissue-committed stem cells and their further differentiation into other tissues comprises muscle cells transdifferentiating into blood cells (Jackson et al. 1999), neural stem cells – into blood cells (Bjornson et al. 1999) and into other tissues (Clarke et al. 2000), HSCs – into neural cells (Eglitis & Mezey 1997, Mezey et al. 2000, Kopen et al. 1999), muscle cells (Gussoni et al. 1999) and hepatocytes (Petersen et al. 1999, Theise et al. 2000a) and bone marrow stromal cells – into neurons (Woodbury et al. 2000). Some comments suggest that cases of ‘transdifferentiation’ might result from cell fusion (Wurmser & Gage 2002, Scott 2004, Kucia et al. 2005), although in other cases fusion was excluded as the cause of observed transdifferentiation (Jang et al. 2004, Wurmser et al. 2004). For example, cases of transdifferentiation of bone marrow cells into liver cells (Alison et al. 2000, Theise et al. 2000b) need to be revised in the light of new experiments, such as fusion in vivo between bone marrow and liver cells (Wang et al. 2003, Camargo et al. 2004) and between bone marrow-derived cells and neural or heart cells (Alvarez-Dolado et al. 2003).

Experimental induction of cell fusion revealed that hybrid cells show phenotype, protein expression profile and differentiation abilities characteristic for less differentiated fusion partner (adult thymocytes with ESCs, Tada et al. 2001; ESCs with bone marrow-derived cells, Terada et al. 2002; ESCs and fibroblasts, Cowan et al. 2005; foetal neural stem cells with ESCs, Ying et al. 2002; different types of somatic cells with ESCs, teratocarcinoma cells, Atsumi et al. 1982, Forejt et al. 1984 and blood stem cells, Camargo et al. 2004, Alvarez-Dolado et al. 2003). In all these cases, the more differentiated cell was reprogrammed (or at least repressed) by less differentiated fusion partner (see also comment by Surani 1999).

Analyses of hybrids between EGCs and thymocytes show the direction of changes in hybrid cells, their level of methylation and phenotype is similar to that of ESCs (Tada et al. 1997). Genome-wide demethylation is characteristic for both PGCs and preimplantation embryonic cells (Monk et al. 1987, Kafri et al. 1992, Ueda et al. 1992).

In our experiments, descendants of FFs were found also in the organs derived from ectoderm and endoderm, which indicates that FFs derived from mesoderm underwent considerable reprogramming. The less differentiated fusion partners for FFs might have been primordial germ cells (PGCs), rare at this stage of development, or cells of early epiblast. These cells do not exhibit totipotency, but still they are less differentiated than FFs.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by grants no. 3P06D 02224 (J A M& A P) and PBZ-KBN-084/P06/2002 (J K) from the State Committee for Scientific Research. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 27 April 2006
First decision 12 June 2006
Revised manuscript received 31 July 2006
Accepted 31 October 2006


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

Alison MR, Poulsom R, Jeffery R, Dhillon AP, Quaglia A, Jacob J, Novelli M, Prentice G, Williamson J & Wright NA 2000 Hepatocytes from non-hepatic adult stem cells. Nature 406 257.[CrossRef][Medline]

Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ & Alvarez-Buylla A 2003 Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 425 968–973.[CrossRef][Medline]

Atsumi T, Shirayoshi Y, Takeichi M & Okada TS 1982 Nullipotent teratocarcinoma cells acquire the pluripotency for differentiation by fusion with somatic cells. Differentiation 23 83–86.[CrossRef][ISI][Medline]

Balinsky D, Spannaus DJ, Yang HY, Briggs CJ, Meyer TE & Warner CM 1983 Unusual glucose phosphate isomerase isozyme patterns in lymphocytes from two C57BL/6J in equilibrium A/J allophenic mice. Journal of Experimental Zoology 227 57–61.[CrossRef][ISI][Medline]

Beddington RS & Robertson EJ 1989 An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 105 733–737.[Abstract/Free Full Text]

Bjornson CR, Rietze RL, Reynolds BA, Magli MC & Vescovi AL 1999 Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 283 534–537.[Abstract/Free Full Text]

Brinster RL 1974 The effect of cells transferred into the mouse blastocyst on subsequent development. Journal of Experimental Medicine 140 1049–1056.[Abstract]

Buehr M & McLaren A 1985 Expression of glucose-phosphate isomerase in relation to growth of the mouse oocyte in vivo and in vitro. Gamete Research 11 271–281.[CrossRef][ISI]

Burnside AS & Collas P 2002 Induction of Oct-3/4 expression in somatic cells by gap junction-mediated cAMP signaling from blastomeres. European Journal of Cell Biology 81 585–591.[CrossRef][ISI][Medline]

Camargo FD, Finegold M & Goodell MA 2004 Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners. Journal of Clinical Investigation 113 1266–1270.[CrossRef][ISI][Medline]

Chapman VM, Ansell JD & McLaren A 1972 Trophoblast giant cell differentiation in the mouse: expression of glucose phosphate isomerase (GPI-1) electrophoretic variants in transferred and chimeric embryos. Developmental Biology 29 48–54.[CrossRef][ISI][Medline]

Clarke DL, Johansson CB, Wilbertz J, Veress B, Nilsson E, Karlstrom H, Lendahl U & Frisen J 2000 Generalized potential of adult neural stem cells. Science 288 1660–1663.[Abstract/Free Full Text]

Cowan CA, Atienza J, Melton DA & Eggan K 2005 Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309 1369–1373.[Abstract/Free Full Text]

DeLorenzo RJ & Ruddle FH 1969 Genetic control of two electrophoretic variants of glucose phosphate isomerase in the mouse (Mus musculus). Biochemical Genetics 3 151–162.[CrossRef][ISI][Medline]

Eakin GS, Hadjantonakis AK, Papaioannou VE & Behringer RR 2005 Developmental potential and behavior of tetraploid cells in the mouse embryo. Developmental Biology 288 150–159.[CrossRef][ISI][Medline]

Eglitis MA & Mezey E 1997 Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. PNAS 94 4080–4085.[Abstract/Free Full Text]

Evans MJ & Kaufman MH 1981 Establishment in culture of pluripotential cells from mouse embryos. Nature 292 154–156.[CrossRef][Medline]

Forejt J, Gregorova S, Dohnal K & Nosek J 1984 Gene expression of differentiated parent in teratocarcinoma cell hybrids. Repression or reprogramming?. Cell Differentiation 15 229–234.[CrossRef][ISI][Medline]

Freshney IR 1987 Culture of Animal Cells. A Manual of Basic Technique, New York: Chichester.

Fulton BP & Whittingham DG 1978 Activation of mammalian oocytes by intracellular injection of calcium. Nature 273 149–151.[CrossRef][Medline]

Gearhart JD & Mintz B 1972a Glucose phosphate isomerase subunit-reassociation tests for maternal–fetal and fetal–fetal cell fusion in the mouse placenta. Developmental Biology 29 55–64.[CrossRef][ISI][Medline]

Gearhart JD & Mintz B 1972b Clonal origins of somites and their muscle derivatives: evidence from allophenic mice. Developmental Biology 29 27–37.[CrossRef][ISI][Medline]

Geiger H, Sick S, Bonifer C & Muller AM 1998 Globin gene expression is reprogrammed in chimeras generated by injecting adult hematopoietic stem cells into mouse blastocysts. Cell 93 1055–1065.[CrossRef][ISI][Medline]

Goto Y, Matsui J & Takagi N 2002 Developmental potential of mouse tetraploid cells in diploid–tetraploid chimeric embryos. International Journal of Developmental Biology 46 741–745.[ISI][Medline]

Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM & Mulligan RC 1999 Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401 390–394.[CrossRef][Medline]

Hakelien AM, Landsverk HB, Robl JM, Skalhegg BS & Collas P 2002 Reprogramming fibroblasts to express T-cell functions using cell extracts. Nature Biotechnology 20 460–466.[CrossRef][ISI][Medline]

Harder F, Henschler R, Junghahn I, Lamers MC & Muller AM 2002 Human hematopoiesis in murine embryos after injecting human cord blood-derived hematopoietic stem cells into murine blastocysts. Blood 99 719–721.[Abstract/Free Full Text]

Hernandez L, Kozlov S, Piras G & Stewart CL 2003 Paternal and maternal genomes confer opposite effects on proliferation, cell-cycle length, senescence, and tumor formation. PNAS 100 13344–13349.[Abstract/Free Full Text]

Horan PK & Slezak SE 1989 Stable cell membrane labelling. Nature 340 167–168.[CrossRef][Medline]

Illmensee K & Mintz B 1976 Totipotency and normal differentiation of single teratocarcinoma cells cloned by injection into blastocysts. PNAS 73 549–553.[Abstract/Free Full Text]

Jackson KA, Mi T & Goodell MA 1999 Hematopoietic potential of stem cells isolated from murine skeletal muscle. PNAS 96 14482–14486.[Abstract/Free Full Text]

Jang YY, Collector MI, Baylin SB, Diehl AM & Sharkis SJ 2004 Hematopoietic stem cells convert into liver cells within days without fusion. Nature Cell Biology 6 532–539.[CrossRef][ISI][Medline]

Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA & Verfaillie CM 2002 Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418 41–49.[CrossRef][Medline]

Kafri T, Ariel M, Brandeis M, Shemer R, Urven L, McCarrey J, Cedar H & Razin A 1992 Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes and Development 6 705–714.[Abstract/Free Full Text]

Kaufman MH 1992 The Atlas of Mouse Development, London, San Diego: Academic Press Limited.

Kaufman MH & Webb S 1990 Postimplantation development of tetraploid mouse embryos produced by electrofusion. Development 110 1121–1132.[Abstract/Free Full Text]

Kopen GC, Prockop DJ & Phinney DG 1999 Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. PNAS 96 10711–10716.[Abstract/Free Full Text]

Kucia M, Ratajczak J & Ratajczak MZ 2005 Are bone marrow stem cells plastic or heterogenous-that is the question. Experimental Hematology 33 613–623.[CrossRef][ISI][Medline]

Kues WA, Petersen B, Mysegades W, Carnwath JW & Niemann H 2005 Isolation of murine and porcine fetal stem cells from somatic tissue. Biology of Reproduction 72 1020–1028.[Abstract/Free Full Text]

Lehtonen E 1980 Changes in cell dimensions and intercellular contacts during cleavage-stage cell cycles in mouse embryonic cells. Journal of Embryology and Experimental Morphology 58 231–249.[ISI][Medline]

Lyon MF & Searle AG 1989 Genetic Variants and Strains of the Laboratory Mouse, Oxford, New York: Oxford University Press.

MacQueen HA & Johnson MH 1983 The fifth cell cycle of the mouse embryo is longer for smaller cells than for larger cells. Journal of Embryology and Experimental Morphology 77 297–308.[ISI][Medline]

Martin GR 1981 Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. PNAS 78 7634–7638.[Abstract/Free Full Text]

McLaren A 1984 Germ cell lineages. In Chimeras in Mouse Developmental Biology, pp 111–129. Ed. A McLaren. London: Academic Press.

Mezey E, Chandross KJ, Harta G, Maki RA & McKercher SR 2000 Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 290 1779–1782.[Abstract/Free Full Text]

Modlinski JA, Reed MA, Wagner TE & Karasiewicz J 1996 Embryonic stem cells: developmental capabilities and their possible use in mammalian embryo cloning. Animal Reproduction Science 42 437–446.[CrossRef][ISI]

Monk M, Boubelik M & Lehnert S 1987 Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99 371–382.[Abstract]

Nagy A, Gocza E, Diaz EM, Prideaux VR, Ivanyi E, Markkula M & Rossant J 1990 Embryonic stem cells alone are able to support fetal development in the mouse. Development 110 815–821.[Abstract/Free Full Text]

Nagy A, Rossant J, Nagy R, Abramow-Newerly W & Roder JC 1993 Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. PNAS 90 8424–8428.[Abstract/Free Full Text]

Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS & Goff JP 1999 Bone marrow as a potential source of hepatic oval cells. Science 284 1168–1170.[Abstract/Free Full Text]

Petzoldt U 1991 Developmental profile of glucose phosphate isomerase allozymes in parthenogenetic and tetraploid mouse embryos. Development 112 471–476.[Abstract]

Prather RS & First NL 1986 Reprograming of murine blastocoele formation. Journal of Experimental Zoology 237 347–350.[CrossRef][ISI][Medline]

Rieske P, Krynska B & Azizi SA 2005 Human fibroblast-derived cell lines have characteristics of embryonic stem cells and cells of neuroectodermal origin. Differentiation 73 474–483.[CrossRef][ISI][Medline]

Robertson EJ 1987 Embryo derived stem cell lines. In Teratocarcinomas and Embryonic Stem Cells. A Practical Approach, pp 71–112. Ed. EJ Robertson. Oxford, Washington DC: IRL Press.

Saburi S, Azuma S, Sato E, Toyoda Y & Tachi C 1997 Developmental fate of single embryonic stem cells microinjected into 8-cell-stage mouse embryos. Differentiation 62 1–11.[CrossRef][ISI][Medline]

Scott EW 2004 Stem cell plasticity or fusion: two approaches to targeted cell therapy. Blood Cells and Molecular Disease 32 65–67.

Snow MHL 1975 Embryonic development of tetraploid mice during the second half of gestation. Journal of Embryology and Experimental Morphology 34 707–721.[ISI][Medline]

Snow MHL 1978 Proliferative centres in embryonic development. In Development in Mammals, Ed. MH Johnson. Amsterdam, New York: Elsevier/North-Holland.

Stewart CL 1993 Production of chimeras between embryonic stem cells and embryos. Methods in Enzymology 225 823–855.[ISI][Medline]

Suemori H, Kadodawa Y, Goto K, Araki I, Kondoh H & Nakatsuji N 1990 A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous beta-galactosidase expression. Cell Differentiation and Development 29 181–186.[CrossRef][ISI][Medline]

Surani MA 1999 Reprogramming a somatic nucleus by trans-modification activity in germ cells. Seminars in Cell and Developmental Biology 10 273–277.

Suwinska A, Ozdzenski W, Waksmundzka M & Tarkowski AK 2005 Experimentally produced diploid-triploid mouse chimaeras develop up to adulthood. Molecular Reproduction and Development 72 362–376.[CrossRef][ISI][Medline]

Tada M, Tada T, Lefebvre L, Barton SC & Surani MA 1997 Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells. EMBO Journal 16 6510–6520.[CrossRef][ISI][Medline]

Tada M, Takahama Y, Abe K, Nakatsuji N & Tada T 2001 Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Current Biology 11 1553–1558.[CrossRef][ISI][Medline]

Tarkowski AK 1961 Mouse chimaeras developed from fused eggs. Nature 190 857–860.[CrossRef][Medline]

Tarkowski AK, Witkowska A & Opas J 1977 Development of cytochalasin B-induced tetraploid and diploid–tetraploid mosaic mouse embryos. Journal of Embryology and Experimental Morphology 41 47–64.[ISI][Medline]

Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE & Scott EW 2002 Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 416 542–545.[CrossRef][Medline]

Theise ND, Badve S, Saxena R, Henegariu O, Sell S, Crawford JM & Krause DS 2000a Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 31 235–240.[CrossRef][ISI][Medline]

Theise ND, Nimmakayalu M, Gardner R, Illei PB, Morgan G, Teperman L, Henegariu O & Krause DS 2000b Liver from bone marrow in humans. Hepatology 32 11–16.[CrossRef][ISI][Medline]

Tokunaga T & Tsunoda Y 1992 Efficacious production of viable germ-line chimera between embryonic stem (ES) cells and 8-cell stage embryos. Development Growth and Differentiation 34 561–566.[CrossRef]

Ueda T, Yamazaki K, Suzuki R, Fujimoto H, Sasaki H, Sakaki Y & Higashinakagawa T 1992 Parental methylation patterns of a transgenic locus in adult somatic tissues are imprinted during gametogenesis. Development 116 831–839.[Abstract]

Wakayama T, Perry AC, Zuccotti M, Johnson KR & Yanagimachi R 1998 Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394 369–374.[CrossRef][Medline]

Wang ZQ, Kiefer F, Urbanek P & Wagner EF 1997 Generation of completely embryonic stem cell-derived mutant mice using tetraploid blastocyst injection. Mechanisms of Development 62 137–145.[CrossRef][ISI][Medline]

Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, Lagasse E, Finegold M, Olson S & Grompe M 2003 Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422 897–901.[CrossRef][Medline]

West JD & Flockhart JH 1994 Genotypically unbalanced diploid–diploid foetal mouse chimaeras: possible relevance to human confined mosaicism. Genetic Research 63 87–99. Erratum in: Genetic Research 1994 63 236.

Wilmut I, Schnieke AE, McWhir J, Kind AJ & Campbell KH 1997 Viable offspring derived from fetal and adult mammalian cells. Nature 385 810–813.[CrossRef][Medline]

Woodbury D, Schwarz EJ, Prockop DJ & Black IB 2000 Adult rat and human bone marrow stromal cells differentiate into neurons. Journal of Neuroscience Research 61 364–370.[CrossRef][ISI][Medline]

Wurmser AE & Gage FH 2002 Stem cells: cell fusion causes confusion. Nature 416 485–487.[CrossRef][Medline]

Wurmser AE, Nakashima K, Summers RG, Toni N, D’Amour KA, Lie DC & Gage FH 2004 Cell fusion-independent differentiation of neural stem cells to the endothelial lineage. Nature 430 350–356.[CrossRef][Medline]

Ying QL, Nichols J, Evans EP & Smith AG 2002 Changing potency by spontaneous fusion. Nature 416 545–548.[CrossRef][Medline]



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