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Embryo |
a Centre for Genome Research, University of Edinburgh, Edinburgh EH9 3JQ, United Kingdom
b Stem Cell Sciences Ltd., Elsternwick, Victoria 3185, Australia
c Walter and Eliza Hall Institute, Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia
d Medical Radiology, University of Edinburgh, Edinburgh EH8 9AG, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ES cells are derived from cultured mouse epiblasts [9–11]. Intact blastocysts or isolated ICMs/epiblasts are cultured on a feeder layer or in a medium containing the cytokine leukemia inhibitory factor (LIF) [12, 13] and allowed to attach to the substrate and outgrow for several days. The resulting cell masses are then disaggregated and plated under the same conditions. Within a few days, colonies of ES cells may appear, which can be picked and cultured further. Protocols for the derivation of ES cells from mouse strain 129 are well established [14, 15]. Even in experienced hands, however, only about 30% of strain 129 blastocysts cultured according to standard derivation procedures will give rise to ES cells [9, 14]. This procedure does not give equally successful results with blastocysts of different mouse strains [16]. Most existing ES cell lines are from the 129 and C57BL/6 strains. It has proven difficult to derive ES cell lines from strains such as CBA (although derivation has been achieved with the use of specialized protocols [9]), whereas other strains such as FVB and NOD are highly refractory.
Considerable effort has been expended to derive ES cells from animals other than the mouse, and there are many reports of cell lines derived from blastocysts of a wide variety of species. However, these cells have rarely been shown to have significant differentiation potential, and there have been no substantiated reports of chimeras generated from blastocyst-derived cells of any species apart from the mouse [17, 18]. Iannaccone et al. [19] reported rat chimeras derived from "rat ES cells", but further analysis revealed that these animals were in fact rat-mouse chimeras [20, 21].
The failure to produce true ES cells from an animal as closely related to the mouse as the rat is puzzling. There are indications, however, of subtle differences between the pluripotent cells of the early embryo of the two species. Epiblasts isolated from rat embryos at late egg cylinder stages are capable of regenerating parietal endoderm [22], a capacity the mouse epiblast loses at the blastocyst stage [23]. It has so far been impossible to generate teratocarcinomas or isolate embryonal carcinoma cells from rat epiblasts or genital ridges, although such tumors and cell lines are readily derived from mouse material.
The inability to derive pluripotent cell lines when rat blastocysts are cultured according to a standard ES cell derivation protocol is not understood and suggests that although the derivation of ES cells from certain mouse strains is relatively straightforward the maintenance of pluripotency during derivation is a complex process. Given the prime role of Oct-4 in maintaining pluripotent status, continuous expression of Oct-4 through the initial stages of derivation may be necessary if pluripotent cell lines are to be established. Two separate enhancer elements have been identified upstream of the Oct-4 gene [24]. An enhancer distal to the promoter directs expression in the preimplantation embryo, primordial germ cells (PGCs), and the ES and EG cell lines, and a proximal enhancer is responsible for expression in the egg cylinder epiblast and P19 EC cells. However, the regulation of Oct-4 expression during attempted cell line derivation has not been described.
We generated transgenic rats in which the reporter/selection marker ß-geo is expressed under the control of the Oct-4 promoter and enhancers. Transgenic embryos were used to track the expression of Oct-4 and to evaluate the possibility of isolating rat ES cells by means of selection for Oct-4-expressing cells. Both the transgene and endogenous Oct-4 are rapidly downregulated in cultured blastocysts. This finding highlights a major barrier to pluripotent cell line derivation from both mouse and rat embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most of the rats used in this project were of the inbred Fischer (F344) strain, although some observations were made on material from DA and Sprague-Dawley blastocyst outgrowths. Except where noted, the mice used were of the inbred 129/Ola and CBA/CA strains. Humane research use of animals was carried out under project licence PPL60/2300 issued by the U.K. Home Office.
Oct-4 ß-Geo Transgenic Rats
Transgenic Fischer rats were generated by pronuclear injection of a transgene consisting of the ß-geo construct (which combines a neomycin-resistance gene with a lacZ reporter [25]) under the control of 3.6 kilobases of the mouse Oct-4 upstream sequence including both proximal and distal enhancers [24]. Forty-one weaned rats were obtained from 460 injected embryos, and from these, 5 founders with an intact ß-geo sequence were identified. One of these animals produced offspring in which the expression of the lacZ reporter reflected the expression pattern of Oct-4 throughout pre- and postimplantation stages of the mouse. A homozygous line of these Oct-4 ß-geo rats was established and had normal viability and fertility.
Feeder Layers
Feeder layers were prepared from gamma-irradiated DIA-M (differentiation inhibitory activity, matrix-bound form) cells. These C3H 10T1/2 fibroblasts are stably transfected with an expression vector for the matrix-associated form of LIF [26]. These feeder layers successfully support the derivation and propagation of mouse strain 129 ES cell lines.
Medium
Blastocysts and cells were cultured in a Glasgow modification of minimal essential medium (Sigma, St. Louis, MO) supplemented with 15% fetal calf serum, penicillin-streptomycin, and human DIA/LIF (prepared in house) [15] at a concentration of 200 U/ml. In some experiments on rat cells, rat LIF [27] was used, with comparable results. EHS cell attachment matrix (Promega, Madison, WI) was added at a concentration of 140 µl/ml. In stem cell selection cultures, G418 was added to the medium at a concentration of 75 or 100 µg/ml.
Culture Protocol
The procedure for the derivation of rat blastocyst-derived cell lines was based on a previously reported method for the derivation of mouse ES cells [14]. Rat blastocysts from spontaneous matings were collected at 4.5 days postcoitum (dpc), the zona pellucida was removed with acid Tyrode medium, and the embryos were transferred to culture medium on a feeder layer of DIA-M cells in 15-mm wells. Cells were allowed to attach and outgrow for 4 days and were then mechanically disaggregated into small clumps and moved to fresh DIA-M feeders in culture medium. Colonies of morphologically undifferentiated cells were picked daily, roughly disaggregated by trituration, and moved to fresh feeders. A similar procedure was used to derive mouse cell lines, although in this case blastocysts were collected at 3.5 dpc, allowed to outgrow in culture for 5 days, and disaggregated in 0.025% trypsin.
This protocol supports the derivation of mouse ES cell lines from strain 129 blastocysts, although there are two significant differences from culture conditions used elsewhere [14, 15]: we included EHS cell attachment matrix in the medium, and we used DIA-M cells as feeders.
Immunosurgery
Immunosurgery was performed on 4.5-dpc rat blastocysts as described by Solter and Knowles [28] using anti-rat whole serum (Sigma) as the antibody and whole rat serum as the source of complement. Immunosurgically isolated ICMs were cultured as described above but were allowed to outgrow for 5–6 days before the first disaggregation.
Staining for ß-Galactosidase Activity
Embryos and blastocyst outgrowths were stained for ß-galactosidase activity according to the method of Hogan et al. [29].
Alkaline Phosphatase and SSEA-1 Staining
Alkaline phosphatase expression was determined by staining with a diagnostic kit for alkaline phosphatase activity in leukocytes (Sigma). The method outlined by Solter and Knowles [30] was used to stain cells for SSEA-1 (stage-specific embryonic antigen). The primary antibody was the monoclonal antibody MC-480, developed by Davor Solter and obtained from the Developmental Studies Hybridoma Bank (Johns Hopkins School of Medicine and University of Iowa) under contract NO1-HD-2-31244 from the NICHD.
In Situ Hybridization
Whole-mount in situ hybridization of cells was carried out according to the protocol of Rosen and Beddington [31]. The cells were fixed and processed in the wells in which they had grown, and the prehybridization step was allowed to proceed overnight. Digoxigenin-labeled antisense riboprobes were synthesised on templates of mouse cDNA sequences. Sense probes were also synthesized for use as controls, and all cultures tested with these were negative. The following mouse cDNA sequences were used.
Oct-4 The template was a StuI fragment corresponding to nucleotides 951–489 of GenBank accession no. X52437 [1]. In embryo whole mounts, this probe hybridizes with rat RNA exclusively in cells expected to express Oct-4, such as the ICM, epiblast, and primordial germ cells. The hybridization signal obtained with rat embryos is strong and comparable in intensity to that observed in mouse embryos.
H19 The H19 probe was described by Poirier et al. [32].
SPARC The full-length coding sequence of SPARC (osteonectin) was used (supplied by Ian Chambers [Centre for Genome Research, University of Edinburgh, Scotland]).
cdx-2 The cdx-2 cDNA was supplied by Hitoshi Niwa (Riken Center for Developmental Biology, Kobe, Japan) and was derived from nucleotides 257–1199 of GenBank accession no. 57420.
Alphafetoprotein Alphafetoprotein cDNA was kindly supplied by Philippe Gabant (Laboratoire du Biologie du Devellopement, Universite Libre de Bruxelles, Gosselie, Belgium).
Cyclin D3 Cyclin D3 cDNA was kindly supplied by Pierre Savatier [33].
Reverse Transcription-Polymerase Chain Reaction
Cells were collected in PBS, and total RNA was prepared using an RNeasy Mini kit (Qiagen, Valencia, CA). Reverse transcription polymerase chain reaction (RT-PCR) was carried out on the RNA with a Superscript First Strand Synthesis system (Gibco BRL, Grand Island, NY) using random hexamers. The cDNA obtained was quantitatively analyzed with LightCycler FastStart DNA Master SYBR Green 1 (Roche, GMBH Mannheim, Germany). For the determination of expression levels of Oct-4, amplification was carried out in the first instance as described by Vassilieva et al. [34] in which primers for mouse Oct-4 were used. Subsequently, new oligos were designed specifically for the rat material, and two separate PCRs were performed using primers for Oct-4 and ß-actin using the Quantitative LightCycler FastStart DNA Master SYBR Green 1 system. Feeder cells and deionized water were included as blanks. The primers used for rat Oct-4 were 5'-AGGAAGCCGACAACAATGAG-3' and 5'-CTGATTGGCGATGTGAGTGA-3'. For rat ß-actin, the primers were 5'-CACTGGCATTGTATGGACT-3' and 5'-ACGGATGTCAACGTCACACT-3'.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The inclusion of a lacZ construct in the ß-geo transgene allowed us to trace the expression of the transgene through various stages of embryogenesis by staining embryos for ß-galactosidase. Transgene expression was readily evident in all cells of the morula and the early blastocyst. In the expanded blastocyst, expression was downregulated in the trophectoderm but remained high in the ICM (Fig. 1A). Expression was maintained in the epiblast of the egg cylinder (Fig. 1B) but was downregulated progressively as gastrulation proceeded. There was then a period of 2–3 days when no staining could be seen in any part of transgenic embryos. However, at 10–11 dpc faint staining was seen in a punctate distribution in the hindgut region, suggestive of re-expression in the PGCs, and subsequently staining was strong in cells of typical PGC appearance and distribution (Fig. 1C). We therefore concluded that apart from a brief hiatus in expression immediately after gastrulation, which may be associated with the transition between enhancers that occurs at this time [23], the pattern of expression of the transgene in vivo accurately reflects the expected expression pattern of Oct-4 in the rat embryo.
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When blastocysts from transgenic rats were cultured according to the culture protocol described above and stained for lacZ activity, staining was readily detectable in the ICM of blastocysts in culture for up to 36 h. After that, although the size of the outgrowth increased considerably, the number of positive cells rapidly decreased until after 4–5 days of culture few or no stained cells remained (Fig. 2, A and B).
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Application of the Oct-4 Selection Strategy to the Derivation of Rat ES Cells
Previously we presented evidence that selection for Oct-4 expression in ES cell cultures can reduce differentiation and enhance self-renewal of stem cells [35]. This finding suggests that pluripotent cells may be induced to differentiate by the presence of differentiated progeny. It has also been reported that selection for Oct-4 transgene-expressing cells in cultures of blastocyst-derived cells can improve the efficiency of ES cell derivation [36]. Embryos from the Oct-4 ß-geo transgenic rats allowed us to test the efficacy of this method in deriving cell lines from rat embryos.
The inclusion of the neomycin analogue G418 in the culture medium is expected to remove differentiated cells but allow Oct-4-expressing cells to remain. Consistent with this expectation, when transgenic blastocysts or isolated ICMs are cultured under G418 selection (75–100 µg/ml G418) most cells rapidly die. Small colonies of morphologically undifferentiated cells can, however, persist in culture for up to three passages or for 2–3 wk. Their proliferation is very slow, and cells on the surface of the outgrowths continually differentiate and die. Over time, this process results in the differentiation and loss of all cells, and none survive culture for >3 wk. Thus, removal of differentiated cells from cultures of rat embryo cells probably is in itself not sufficient to inhibit differentiation of pluripotent cells or to maintain stem cells in a continuous state of self-renewal. The selection approach does, however, have some positive effect on the survival of undifferentiated cells in the presence of G418 because it allows these cells to persist for 2–3 wk, whereas all cells from nontransgenic blastocysts are eliminated within a few days.
Pattern of Oct-4 Expression in Outgrowths of Nontransgenic Rat and Mouse Blastocysts
Examination of blastocyst outgrowths from Oct-4 ß-geo transgenic rats revealed that transgene expression was rapidly downregulated during the culture period (Fig. 2, A and B). To determine whether this pattern reflected a downregulation of the endogenous Oct-4 gene or was due to anomalous expression of the transgene, we performed whole-mount in situ hybridization with blastocyst outgrowths of nontransgenic rats from the early outgrowth stage until after the first disaggregation. The pattern of Oct-4 mRNA as determined by in situ hybridization was identical to that observed after ß-galactosidase staining in the transgenic rats. Thus, expression of endogenous Oct-4 is not maintained in culture.
To determine whether the loss of Oct-4 was specific to the rat, we examined the expression of Oct-4 in outgrowths of blastocysts from several mouse strains (C57BL/6, 129, MF1, and CBA). Downregulation of Oct-4 occurred in the same pattern in rat and mouse, and the following description applies to both species and all strains examined.
Approximately 36 h after a blastocyst culture was begun, the embryo attached to the substrate and the cells of the trophectoderm began to outgrow. At this stage, the cells of the epiblast clearly expressed Oct-4 (Fig. 3A). After 3–4 days of culture, however, most of the cells in the mass of the outgrowth were negative for Oct-4, and expression was confined to a small number of cells in the center (Fig. 3B). By 5 days of culture, the cell mass had grown considerably in size, but the proportion of Oct-4-expressing cells appeared to have decreased (Fig. 3C).
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In a few mouse outgrowths (
1% of cultures), patches of Oct-4-positive cells were seen growing out on the substrate. In most cases, however, the population of Oct-4-expressing cells did not increase during blastocyst outgrowth but rather diminished and often disappeared entirely after 5–6 days.
Positive and negative cells within the cell mass were morphologically indistinguishable. Both appeared undifferentiated, and it was impossible to discriminate between them in unstained cultures. Thus, although morphologically undifferentiated cells proliferated during the outgrowth stage, few or none of these cells retained Oct-4 expression for more than 5–6 days.
This general sequence of events was observed consistently in blastocyst outgrowths of DA, Sprague-Dawley, and transgenic and nontransgenic Fischer rats and C57BL/6, 129, MF1, and CBA mice. The pattern of loss of expression of Oct-4 in blastocyst outgrowths was then studied in more detail in cultures of strain 129 and CBA blastocysts.
Retention of Oct-4-Expressing Cells in Strain 129 and CBA Outgrowths
As blastocysts initially attached and outgrew (3–4 days of culture), cells expressing Oct-4 were present in almost all outgrowths. By 5 days, however, the number of outgrowths containing positive cells began to drop. This effect was more pronounced in cultures of CBA than in those of strain 129 blastocysts. By 6 days of culture, only 4% of CBA outgrowths retained expressing cells compared with 44% of strain 129 outgrowths (Table 1).
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After outgrowths were disaggregated and replated, no expression of Oct-4 was ever seen in cultures of rat or CBA cells. In contrast, disaggregated strain 129 outgrowths frequently gave rise to colonies of Oct-4-positive cells with the distinctive morphology of ES cells. Colonies that resembled ES cells but did not express Oct-4 were not seen nor did any differentiated cells express Oct-4.
The efficiency of ES cell derivation can be improved if isolated ICMs, rather than intact blastocysts, are cultured and disaggregated [9]. We therefore examined the pattern of Oct-4 expression in outgrowths of immunosurgically isolated ICMs of mouse strain 129 and CBA embryos and rat embryos. The results were similar in all groups. After 5–6 days in culture, approximately half of ICM outgrowths retained a strong, uniform Oct-4 expression, whereas the remainder had lost Oct-4 expression entirely or expression was faint or patchy (Fig. 4, A and B). Oct-4-negative cells in such morphologically undifferentiated ICM outgrowths are unlikely to be accounted for by trophectoderm. Because every cell in an isolated ICM should express Oct-4 [37], this observation suggests that Oct-4 expression in cells of the ICM can undergo downregulation in culture.
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Culture of Blastocyst-Derived Cells
The mass of Oct-4-negative but morphologically undifferentiated cells remaining after 5 or 6 days of culture could be disaggregated and cultured further. Over several days, all the cells from disaggregated mouse outgrowths differentiated into giant cells and other H19-positive cells of undetermined types. We were unable to maintain cultures of undifferentiated Oct-4-negative cells from mouse blastocyst outgrowths, although colonies of Oct-4-positive ES cells may apppear in cultures derived from strain 129 blastocysts. In contrast, Oct-4-negative cells from rat outgrowths were maintained continuously in a proliferative and morphologically undifferentiated state (Fig. 5A). Cultures of these cells were established from both intact blastocysts and immunosurgically isolated ICMs.
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There are strain differences in the efficiency with which these cells can be obtained. Over 90% of Fischer rat blastocysts (both transgenic and nontransgenic) gave rise to stable lines, but <10% of DA blastocysts yielded lines. We derived >50 lines from Fischer rat embryos and maintained 4 of these for >50 passages (equivalent to >4 mo). Three of these four lines were examined for euploidy at high passage numbers (>35). More than 80% of metaphase plates examined (>50 for each line) demonstrated a euploid complement of 42 chromosomes.
These rat cells resembled mouse ES cells in their general morphology and grew as rounded colonies of small, tightly packed cells that expressed alkaline phosphatase (Fig. 5, A and B). They also expressed SSEA-1, although this marker was expressed patchily, apparently only by cells in the interior of colonies (Fig. 5C).
If maintained for several days without passaging, these cells differentiated spontaneously. Rounded cells expressing SPARC and with the refractile character of parietal endoderm appeared in large numbers. Large H19-positive cells with the flattened morphology characteristic of trophoblast giant cells were also common, as were irregular masses of H19-positive cells whose identity was not clear. On aggregation, the cells did not form embryoid bodies and showed no evidence of germ-layer development but differentiated entirely into H19-positive cells.
These cells were factor and feeder dependent, growing poorly on feeders of primary mouse embryonic fibroblasts and not at all on gelatin. When LIF was removed from the medium, the cells could be passaged five or six times on DIA-M feeders but progressively differentiated. These cells were also dependent on the presence of EHS matrix in the medium; when this matrix was omitted, the cells rapidly differentiated. When colonies were passaged every 2 or 3 days on DIA-M feeders in medium supplemented with both LIF and EHS matrix, they could be maintained in a morphologically undifferentiated state.
In situ hybridization revealed that the undifferentiated cells did not express SPARC or alphafetoprotein (markers of parietal and visceral endoderm, respectively) but did express cdx-2 and cyclin D3 (Fig. 5, D and E), although not uniformly. Both of these markers are found in diploid trophectoderm [33, 38].
Vassilieva et al. [34] derived cells described as "ES-cell like" from rat blastocysts, and these cells were very similar to the cells we isolated. Vassilieva et al. reported that Oct-4 transcripts were detected in their cells by RT-PCR. We therefore carried out a quantitative RT-PCR analysis on our cultures. A low level of Oct-4 mRNA was detectable in one of three lines examined. However, even in this line we were unable to demonstrate Oct-4 transcripts by in situ hybridization, indicating that the levels of expression were not significant. Consistent with this, lines derived from Oct-4 ß-geo transgenic embryos did not show any expression of ß-galactosidase nor were they resistant to G418.
Production of Chimeras from Rat ICMs but Not from Derivative Cell Lines
We also investigated whether the cells could contribute to chimeras. First, we established a technique of generating rat chimeras. Freshly isolated Fischer (albino) ICM cells were injected under the zona pellucida of recipient eight-cell DA (agouti) embryos. Of 17 pups born from transferred embryos, 9 were coat-color chimeras. We then injected cells from our cultured lines (of the Fischer strain) into recipient DA embryos. Cells from four lines were used in separate experiments, two of low passage number (10) and two at passages 20–30. Of the 82 rats born from injected embryos, none showed coat-color chimerism. Microsatellite analysis also failed to show any Fischer contribution to either adult or fetal tissues.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oct-4-positive and Oct-4-negative cells in outgrowths are indistinguishable. Furthermore, loss of Oct-4 occurs in immunosurgically isolated ICMs without extensive cell death or overt differentiation. These observations argue in favor of direct downregulation of Oct-4 rather than selective death of Oct-4-expressing cells or overgrowth by a differentiated lineage and imply that Oct-4-positive and -negative cells represent epiblast before and immediately after loss of pluripotency. When derived from rat outgrowths, these Oct-4-negative cells can be readily maintained as stable cultures. They express the markers cdx-2 and cyclin D3, characteristic of trophectoderm, but do not express Oct-4 as determined by whole-mount in situ hybridization. Vassilieva et al. [34] described rat "ES-cell-like cells" derived in a similar way that appeared to express Oct-4 as determined by RT-PCR. These authors presented no evidence, however, that their ES-cell-like cells had any capacity for differentiation. We performed real-time quantitative RT-PCR on our cells and detected very low levels of Oct-4 transcript in one of three lines tested. However, even this line was not positive when analyzed by in situ hybridization. Thus, any expression of the Oct-4 gene in these cells must be at a very low level. The level of expression is likely to be critical, because a specific level of Oct-4 expression in an ES cell is essential to the maintenance of pluripotency [8]. In spite of the absence of Oct-4, both the cells described here and those of Vassilieva et al. [34] are similar to mouse ES cells in morphology, factor dependence, and expression of alkaline phosphatase and SSEA-1 (although the latter marker is expressed patchily). These findings suggest that some so-called ES-cell-like cell lines derived from blastocysts may represent cells that, although resembling ES cells superficially, lack sufficient Oct-4 expression to maintain pluripotency and are hence incapable of contributing to fetal germ layers.
The rat embryo-derived cells express at least two markers characteristic of trophectoderm derivatives and can spontaneously differentiate into trophoblast giant cells. This finding is consistent with a depletion of Oct-4 leading to diversion of epiblast cells into the trophectoderm lineage, as occurs when Oct-4 is acutely eliminated from ES cells [8]. However, although the cells appear capable of differentiation into trophoblast, they are distinguishable from trophoblast stem (TS) cells [39]. When cultured under conditions appropriate for TS cell derivation, i.e., on feeders in the presence of fibroblast growth factor 4 (FGF-4), rat blastocysts can give rise to continuously proliferating colonies similar in appearance to mouse TS cells. Such TS cells characteristically grow as thin, flattened colonies (Fig. 6), and their propagation is dependent upon high concentrations of FGF-4. In contrast, the cells we have isolated grow as tightly packed clumps and in the absence of exogenous FGF-4. Furthermore, they are capable of generating at least one nontrophectoderm derivative, parietal endoderm. Therefore, they appear to represent a novel class of extraembryonic stem cell.
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Although the rat embryo-derived cells of this study do not possess the potency of ES cells, they may still have practical applications. Because these cells retain diploidy after many passages and are transfectable (unpublished results), they could be used for targeted genetic modification and the nuclei could be transferred into recipient oocytes to generate transgenic rats. Fändrich et al. [40] reported blastocyst-derived rat cell lines with features in common with the cells we isolated. Their cells resembled mouse ES cells morphologically, expressed alkaline phosphatase, and were "mosaic" for SSEA-1 expression. No mention of Oct-4 expression was made nor of differentiation in vitro or generation of embryo chimeras. However, these cells appeared to give rise to hematopoietic derivatives when introduced directly into adult rats.
The present findings call into question the general assumption that ES cell precursors expand during the primary outgrowth stage. If Oct-4 expression is not maintained in primary outgrowths, ES cell precursors are probably lost at this stage. This fact may underlie the difficulties commonly experienced in deriving ES cell lines. Accordingly, the higher frequency and greater longevity of Oct-4 expression in strain 129 outgrowths may contribute to the permissiveness of this strain for ES cell derivation. If, as seems likely, true ES cells are derived directly from the Oct-4-expressing cells of the epiblast, one of the keys to successful derivation may lie in conditions or treatments that allow the maintainance of Oct-4 expression in epiblast derivatives throughout the outgrowth phase and beyond the first disaggregation. To this end, the analysis of Oct-4 expression in blastocyst outgrowths may be used as an assay system to screen factors and protocols for their possible utility in enhancing the efficiency of ES cell derivation. Such studies will be facilitated by the use of transgenic reporters such as the Oct-4 ß-geo construct described here or others such as Oct-4 GFP (green fluorescent protein).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Mia Buehr, Centre for Genome Research, University of Edinburgh, King's Buildings, West Mains Rd., Edinburgh EH9 3JQ, U.K. FAX: 44 131 650 3777; mbuehr{at}srv0.bio.ed.ac.uk
Received: 2 April 2002.
First decision: 30 April 2002.
Accepted: 1 July 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 K. Hochedlinger and R. Jaenisch Nuclear Transplantation, Embryonic Stem Cells, and the Potential for Cell Therapy N. Engl. J. Med., July 17, 2003; 349(3): 275 - 286. [Full Text] [PDF]
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