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Establishment of Male and Female Nuclear Transfer Embryonic Stem Cell Lines from Different Mouse Strains and Tissues¹

Center for Developmental Biology,³ RIKEN Kobe, Kobe 650-0047, Japan Department of Life Science,⁴ Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan Graduate School of Agricultural Science,⁵ Tohoku University, Sendai 981-8555, Japan

	ABSTRACT

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Nuclear transfer can be used to generate embryonic stem cell lines from somatic cells, and these have great potential in regenerative medicine. However, it is still unclear whether any individual or cell type can be used to generate such lines. Here, we tested seven different male and female mouse genotypes and three cell types as sources of nuclei to determine the efficiency of establishing nuclear transfer embryonic stem cell lines. Lines were successfully established from all sources. Cumulus cell nuclei from F₁ mouse genotypes showed a significantly higher cumulative establishment rate from reconstructed oocytes than from other cells; however, there were no genotype differences in success rates from cloned blastocysts. Thus, the overall success depends on preimplantation development, and, once embryos have reached the blastocyst stage, the genotype differences disappear. All mouse genotypes that were tested demonstrated at least one cell line that subsequently contributed to germline transmission in chimeric mice, so these cell lines clearly possess the same potential as embryonic stem cells derived from fertilized embryos. Thus, nuclear transfer embryonic stem cells can be generated relatively easily from a variety of inbred mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly.

assisted reproductive technology, early development, embryo

	INTRODUCTION

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There is much active research into the possible applications of human embryonic stem (ES) cells in regenerative medicine, in which ES cells are envisioned as potential sources for use in cell replacement therapies. However, as with any allogeneic material, ES cells derived from fertilized blastocysts, and the progeny of such cells, inevitably face the risk of immunorejection when transplanted. It has been proposed that ES cells derived from embryos cloned from a host patient's own cells represent a potential solution to the problem of rejection, because any replacement cells would be genetically identical to the host's own [1–5]. Hwang et al. [6] have now successfully derived ES cells from a human embryo cloned from a somatic cell nucleus using nuclear transplantation technology. This in itself does not prove that such cells can be used effectively in regenerative medicine; many challenges remain, including the induction and targeting of differentiation, the control of stem cell proliferation, and the dilemma of persistent re-rejection in cases of autoimmune disease. However, it does provide an important first proof-of-principle of the feasibility of creating ES cells using a patient's own ES cells as a source. However, in this experiment, only one cell line was established, from a woman who provided both oocytes and somatic (cumulus) cell nuclei. It raises the question of whether only healthy women can act as ES cell donors or whether these cells can be derived from any individual.

ES-like cell lines generated from somatic cells via nuclear transfer (NTES cells) were first reported for the cow [7] and then the mouse [8, 9]. These NTES cell lines are believed to possess the same capacities for unlimited differentiation and self-renewal as those of conventional ES cell lines derived from normal embryos produced by fertilization. We have previously shown that NTES cell lines are capable of differentiating into all three germ layers in vitro, or into spermatozoa and oocytes in chimeric mice [10]. This was the first demonstration that NTES cells have the same developmental potential as fertilized ES cells. Moreover, cloned mice can be obtained from these NTES cell lines using a second nuclear transfer [10]. These techniques have now been applied to basic research, such as to demonstrate irreversible changes to DNA in adult lymphocytes [11], but not olfactory neurons [12, 13], and to examine the characteristics of different types of cancer cells [14, 15]. However, NTES cells are not yet fully characterized in terms of the effects of the animal strain genotype or sex of the donor nucleus. Such factors often affect the successful full-term development of cloned animals [16, 17]. For example, the success rate of establishing cloned mice depends on the mouse genotype; hybrid genotypes are more tolerant of cloning than inbred genotypes, such as C57BL/6 and C3H/He, which are in common use in mouse genetic studies, but which have never been cloned successfully [16, 17].

Here, we report that NTES cells can be generated from the tissues of both males and females of different mouse genotypes using different cell types, with higher success rates than for adult somatic cell nuclear cloning.

	MATERIALS AND METHODS

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Animals

The nuclear donor mouse genotypes used were B6D2F₁ (C57BL/6 x DBA/2), B6C3F₁ (C57BL/6 x C3H/He), C57BL/6, C3H/He, DBA/2, and transgenic mouse lines (B6D2F₁ and 129B6F₁ backgrounds) with the green fluorescent protein (GFP) gene inserted [18, 19]. Enucleated B6D2F₁ oocytes were used as recipients for nuclear transfer. In experiments to create chimeras, normally fertilized blastocysts of the BALB/c or ICR strains were used as recipients for NTES cell injections. The surrogate mothers carrying chimeric embryos to term were ICR mice. All animals (obtained from SLC, Shizuoka, Japan) were maintained in accordance with the Animal Experiment Handbook at the Riken Center for Developmental Biology.

Establishment of NTES Cell Lines

Nuclear transfer was performed as described [20]. Cultured tail-tip fibroblasts, freshly isolated cumulus cells, or testicular Sertoli cells were used as nuclear donors from the genotypes described above [10, 20–23]. After nuclear transfer, the reconstructed oocytes were activated by 10 mM SrCl₂ in Ca²⁺-free CZB medium in the presence of 5 g/ml cytochalasin B and cultured for 4 days in KSOM medium (Specialty Media, Lavallette, NJ). When they had developed to the morula or blastocyst stages, they were used to establish NTES cell lines as described [10] with a slight modification, in that 20% Knock-out Serum Replacement (Invitrogen, Carlsbad, CA) plus 0.1 mg/ml ACTH (fragments 1–24; American Peptide Company, Sunnyvale, CA) instead of fetal calf serum was added to the ES cell medium [24]. Briefly, morulae/blastocysts were treated with acid Tyrode solution to remove the zonae pellucidae and placed in 96-multiwell dishes precoated with mouse embryonic fibroblasts for 10 days or more. Proliferating outgrowths were dissociated using trypsin and replated on fibroblasts until stable cell lines grew out. Pluripotency of the established NTES cell lines was determined by alkaline phosphatase staining and from embryoid body formation. In some cell lines, the karyotypes were examined by spectral karyotyping and fluorescent in situ hybridization (SKY-FISH) chromosome painting (Applied Spectral Imaging, Carlsbad, CA) according to the manufacturer's instructions. As controls, we also established ES cell lines from C3H/He, C57BL/6, or DBA/2 fertilized embryos in which morulae or blastocysts were collected from the tracts of normally pregnant mice 4 days after mating, and cultured as above.

Production of Chimeric Mice and Confirmation of Germline Transmission of NTES Cells

The NTES cells (from dark or gray-colored donor mice) were introduced into the blastocoels of (albino) E3.5 BALB/c or ICR strain blastocysts by piezo-assisted microinjection (Primetech Corporation, Tokyo, Japan). Immediately after injection, the blastocysts were transferred into pseudopregnant ICR-strain surrogate mothers [10]. When mature, chimeric offspring showing dark or gray coat colors were selected at random and mated with ICR mice. The chimeric mice constructed using NTES cells derived from Tg-mice expressing GFP were killed and dissected, and any GFP expression in tissues was detected using a UV lamp.

Statistical Analyses

Outcomes were evaluated using chi-square tests, and P < 0.05 was assumed to be statistically significant.

	RESULTS

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To demonstrate that therapeutic cloning techniques can be applied to a range of genotypes, we used three inbred and two hybrid mouse genotypes of both sexes. The C3H strain has an agouti coat color; C57BL/6 is black; and DBA has a dilute brown, nonagouti coat color. Hybrids show both phenotypes of the parental genotypes used. We also used the 3 Tg genotype, which expresses GFP as a cell marker, to test for its contribution to the chimeric mice.

Establishment of NTES Cell Lines

A total of 2999 enucleated oocytes were subjected to nuclear transfer, and 1506 (50.2%) survived nuclear injection and artificial activation. Of those, 472 (31.3%) embryos developed to the morula or blastocyst stage, and 85 NTES cell lines (18.0% from blastocysts, and 5.6% from activated oocytes) were established.

Effects of Donor Cell Genotype on ES Cell Derivation

The overall success rates of pseudopronucleus formation, embryo development, and establishment of NTES cell lines were not significantly different between genotype (Tables 1 and 2). However, when comparing inbred and F₁ genotypes, the blastocyst development and NTES cell derivation rates from reconstructed oocytes were significantly better from F₁ cumulus cells than from others (blastocyst development, 45% vs. 4%–29%; NTES cell derivation, 8% vs. 1%–3%; P < 0.01 by chi-square tests, respectively; Fig. 1a). This difference was observed only when the data were compared from reconstructed oocytes. When we compared the rate of establishment of NTES cell lines from cloned blastocysts (Fig. 1b), there was no significant difference between groups. Thus, the better overall success rate for F₁ cumulus cells was the result of more cells developing to the blastocyst stage after nuclear transfer.

View larger version (34K): [in this window] [in a new window]   FIG. 1. Comparisons of the success rates in establishing NTES cell lines between genotype and sexes. a) Establishment rates were calculated based on the numbers of reconstructed oocytes. When the donor nuclei were from F1 female cumulus cell nuclei, the overall rate was significantly better than rates achieved using other sources. b) Establishment rates were calculated based on the numbers of cloned morulae or blastocysts. There were no statistically significant differences between sources

Effect of Donor Cell Sex on ES Cell Derivation

When we compared the effect of donor cell sex using tail-tip fibroblasts as donor nuclei, there was no significant difference between males and females in any group except that the rate of pseudopronuclear formation in inbred females was inferior (Tables 1 and 2; Fig. 1, a and b).

Effects of Donor Cell Type on ES Cell Derivation

We compared the effect of donor cell type using female cumulus cell nuclei and female tail-tip cell nuclei. Pseudopronucleus formation, embryo development, and NTES cell derivation were all significantly better when F₁ cumulus cells were used for donor nuclei than when tail-tip cells were used (pseudopronucleus formation, 89% vs. 48%–70%; morula or blastocyst development, 45% vs. 4%– 11%; NTES cell derivation, 8% vs. 1%–2%, respectively; P < 0.01). This difference was not observed when inbred genotype cumulus cells were compared with tail-tip cells (Fig. 1, a and b). We also used male Sertoli cells as nuclear donors and found a higher establishment rate, but the data are insufficient to make any conclusion. As a control for our conditions, we also established ES cell lines from fertilized embryos. The rate from fertilized blastocysts was 25%–68% for each genotype, as reported previously [24].

Germline Transmission

All established cell lines were positive for alkaline phosphatase staining and formed embryoid bodies (data not shown), suggesting that these NTES cell lines would have been pluripotent [10]. About 80% of the cells from six tested NTES cell lines (two from hybrid donors and four from inbred donors) had normal karyotypes, as shown by SKY-FISH chromosome painting (data not shown). To test the possible pluripotency of these NTES cells, chimeric mice were produced by injecting the NTES cells into BALB/c or ICR blastocysts and transferring these to pseudopregnant recipients. Chimeric mice with a donor genotype of dark, brown, or gray coat color were obtained from all NTES cell lines. After sexual maturation, the chimeric mice were mated with mice of the ICR strain. At least one chimeric animal of all mouse genotypes delivered nonalbino offspring, demonstrating true germline transmission of the NTES cell genotype (Table 3). When the organs of high-coat-color chimeric mice derived from GFP-expressing NTES cells were examined, all tissues examined (brain, lung, heart, kidney, intestines, liver, and pancreas) exhibited GFP emission under UV illumination (data not shown).

	DISCUSSION

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The main objective of this study was to evaluate the possibility of producing NTES cell lines from several tissues in various male and female mouse genotypes, because for therapeutic cloning, to be practical, one needs to be able to work with a variety of genotypes. We demonstrated that all genotypes, both male and female, could be used to derive NTES cell lines from somatic cell nuclei with a relatively high success rate, even in the case of inbred genotypes such as C57BL/6 and C3H/He. These genotypes have been used for somatic cell nuclear transfer experiments but have never produced clones [16]. Most (80%) of the examined NTES cells we produced here showed normal karyotypes and demonstrated germ line transmission, which confirms their pluripotency. The characteristics of these NTES cells were nearly identical to those of ES cells derived from fertilized embryos and were able to differentiate into spermatozoa or oocytes in the chimeric mice in vivo.

Attaining blastocyst development from cloned embryos is relatively easy in all mouse genotypes and tissues, as previously reported [16, 17], but most of them degenerate during culture or just after implantation [10, 25]. Hochedlinger et al. [15] and Li et al. [26] reported that cloned embryos derived from cancer cell nuclei with abnormal chromosomes could develop to the blastocyst stage. However, most cloned embryos die immediately after implantation if transferred into pseudopregnant uteri [26], and this is believed to be caused either by abnormal chromosomes or through incomplete genomic reprogramming. Moreover, even if some embryonic genes that are essential for full-term development are not expressed in cloned embryo, they still develop to the blastocyst stage [27, 28]. On the other hand, when oocytes were fertilized with karyotypically abnormal spermatozoa produced by freeze-drying, more than half the embryos showed chromosomal abnormalities [29]. Those abnormal embryos could not develop to full term; however, most could develop to morphologically "normal" blastocysts [30]. This suggests that the presence of a normal karyotype or complete genomic reprogramming is not essential for preimplantation development.

In our previous work, the rate of production of NTES cell lines derived from cumulus cell nuclei was 14% from cloned blastocysts, which was significantly higher than from tail-tip cell nuclei (5%), and the overall establishment rate was 8.8% [10]. In the present study, the rate of establishment of NTES cell lines from cumulus cell nuclei was 9%–19%, as before, but that from tail-tip cell nuclei was 22%, and the overall success rate was 18%. This improvement could be explained by better nuclear transfer and NTES cell derivation techniques, such as a newly modified medium [24]. However, different mouse genotypes (either different genotypes or different supplying companies), and possibly increased skill of the experimentalists [31], were involved in this study. Blelloch et al. [14] reported that 44%–57% of cloned blastocysts derived from embryonic carcinoma (EC) and ES cell nuclei could be used successfully to establish NTES cell lines, and suggested that the epigenetic state of the EC/ES cell genomes can be more efficiently reprogrammed than somatic cell genomes. This is a reasonable hypothesis, but we may be able to increase the rate of establishing NTES cell lines from somatic cell nuclei even more by improving and optimizing the nuclear transfer and NTES cell derivation techniques.

The rate of establishment of NTES cell lines from hybrid genotype cumulus cell nuclei was significantly higher than from other donor cell nuclei (8% vs. 1%–3%) using reconstructed oocytes. However, this difference disappeared when the rates were compared from cloned blastocysts (19% vs. 9%–25%). Thus, the overall success rate appears to depend primarily on preimplantation development; once embryos have reached the blastocyst stage, the genotype differences are no longer significant. The tail-tip cells required extensive treatment before use, such as in vitro culture for 2 wk, trypsin treatment, and at least three washes. Moreover, those cells are large with a robust membrane, and it is difficult to isolate and inject the nuclei into enucleated oocytes. On the other hand, the cumulus cells could be collected at the same time as the oocytes and did not need any other treatment before use. Cumulus cells are smaller than fibroblasts and have a weaker cell membrane, so it is easier to isolate the nuclei and inject them into enucleated oocytes. It is therefore possible that the method of donor cell nuclei preparation is important for the successful preimplantation development of reconstructed oocytes. To increase the overall results, we thus need to improve the methods for preparing nuclei for transfer.

Although the success rates using fibroblast nuclei were limited, we showed a relatively high rate of establishment of NTES cell lines from all genotypes tested, including males and females (18% average from blastocyst). Fibroblasts are a ready source of nuclei that will enable us to apply these NTES cell techniques to basic science. For example, if mutant mouse genotypes of interest are infertile and produce no germ cells, this NTES cell technique may be the only way to preserve such genotypes [32]. However, for human therapeutic cloning, further study is required, because we do not yet know whether these NTES cells have the same potential as true ES cells.

	ACKNOWLEDGMENTS

 
We thank Mr. D. Sipp, Dr. H. Niwa, and Dr. J. Cummins for critical and useful comments on the manuscript.

	FOOTNOTES

 
¹ Supported by a Grant-in-Aid for Creative Scientific Research (13GS0008), Scientific Research in Priority Areas (15080211), Young Scientists A (15681014), and a project for the realization of regenerative medicine (the research field for the technical development of stem cell manipulation) to T.W. from the Ministry of Education, Science, Sports, Culture and Technology of Japan.

² Correspondence: Teruhiko Wakayama, 2-2-3 Minatojima-minamimachi Chuo-ku, Kobe 650-0047, Japan. FAX: 81 78 306 0101;teru{at}cdb.riken.go.jp

Received: 13 August 2004.

First decision: 13 September 2004.

Accepted: 2 December 2004.

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