Efficient Establishment of Mouse Embryonic Stem Cell Lines from Single Blastomer - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:55 |只看该作者 |倒序浏览 |打印

作者：Sayaka Wakayama, Takafusa Hikichi, Rinako Suetsugu, Yuko Sakaide, Hong-Thuy Bui, Eiji Mizutani, Teruhiko Wakayama作者单位：Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN Kobe, Kobe, Japan 8 k  C7 V' M. K& M7 o  U' q

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      【摘要】1 Y; p1 e2 [+ ?- f5 K
   Recently, ES cell lines were established from single blastomeres taken from eight-cell embryos in mice and humans with success rates of 4% and 2%, respectively, which suggests that the method could be used in regenerative medicine to reduce ethical concerns over harm to embryos. However, those studies used other ES cells as supporting cells. Here, we report a simple and highly efficient method of establishing mouse ES cell lines from single blastomeres, in which single blastomeres are simply plated onto a feeder layer of mouse embryonic fibroblasts with modified ES cell medium. A total of 112 ES cell lines were established from two-cell (establishment rate, 50%¨C69%), early four-cell (28%¨C40%), late four-cell (22%), and eight-cell (14%¨C16%) stage embryos. We also successfully established 18 parthenogenetic ES cell lines from first (36%¨C40%) and second polar bodies (33%), the nuclei of which were reconstructed to embryos by nuclear transfer. Most cell lines examined maintained normal karyotypes and expressed markers of pluripotency, including germline transmission in chimeric mice. Our results suggest that the single cells of all early-stage embryos or polar bodies have the potential to be converted into ES cells without any special treatment.+ {0 a* @7 B1 z, {
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Disclosure of potential conflicts of interest is found at the end of this article. . p6 e5 p8 ~! \( I
      【关键词】 Embryonic stem cell Blastomere Polar body Nuclear transfer, n  T- N; O) y6 l- h! ?1 u2 [
   INTRODUCTION/ K' C; E( h: q
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There is much research into the applications of human embryonic stem (ES) cells in regenerative medicine, where ES cells are envisioned to be potential resources for cell or tissue replacement therapies. However, the ethical controversy surrounding the derivation of ES cells arises from the need to destroy the embryos. Several approaches that circumvent this problem have recently been reported .2 |3 X* C- ?! K0 T; ~! ]* J

For example, ES cell lines can be established nondestructively from single blastomeres. The developmental capacity of single blastomeres isolated from mammalian embryos has been studied extensively, and it is clear that they retain their pluripotency . However, the overall success rates were only 4% and 2%, respectively (5 ES cell lines from 125 attempts in mice and 2 ES cell lines from 91 attempts in humans). The protocol also requires the use of other ES cell lines to support blastomere development, which may increase the risk of contamination. Nevertheless, this research points to the potential for banking autologous ES cell lines, although the technique clearly needs improvement if it is to be applied to human regenerative medicine. In particular, if it could be used for frozen embryos, which are the surplus embryos remaining after infertility treatments, the number of established ES cell lines should increase significantly, and it should be easy to find immunologically competent ES cell lines in cell banks.

Parthenogenetically activated oocytes could also be used to establish ES cell lines (pES cells). Parthenogenesis is the biological phenomenon in which embryonic development is initiated without male contribution. When the parthenogenetic embryos from mammalian oocytes are transferred into surrogate mothers, they are capable of surviving to day 10 of development in mice, day 21 in sheep, day 29 in pigs, and day 12 in rabbits . If the first and second polar bodies can be used to establish pES cell lines, the destruction of the donor oocyte, like that of the biopsied embryo, can be avoided. Here, we report that mouse ES cell lines can be established easily and efficiently from single blastomeres from embryos or polar bodies without the use of other ES cells.+ P! R; W/ V" x9 R" x

MATERIALS AND METHODS4 d  {+ d& I4 z$ o

Animals

B6D2F1 (C57BL/6 x DBA/2), 129/Sv ICR, and transgenic mouse lines (ICR backgrounds) carrying the gene for green fluorescent protein (GFP) were used . B6D2F1 oocytes were used as the recipients in polar body nuclear transfer. In experiments to create chimeras, normally fertilized blastocysts of BALB/c, ICR, or B6D2F2 strains were used as the recipients of ES cell injections. The surrogate mothers carrying chimeric embryos to term were ICR mice. The ICR strain was used as the source of mouse embryonic feeder cells (MEFs). All animals (obtained from SLC, Shizuoka, Japan) were maintained in accordance with the Animal Experiment Handbook of the RIKEN Center for Developmental Biology, Kobe, Japan.

Establishment of ES Cell Lines from Blastomeres
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B6D2F2 or GFP-expressing fertilized embryos were collected from female B6D2F1 mice mated with B6D2F1 or GFP-ICR males, respectively, at approximately 40 hours after the injection of human chorionic gonadotropin and were incubated at 37¡ãC under 5% CO2 in air until use. Oocytes and zygotes were collected the following morning from female B6D2F1 or 129/Sv mice mated or not with 129/Sv males. Single cells were removed from two-cell, early four-cell, late four-cell, and eight-cell stage mouse embryos at 40, 48, 62, and 64 hours, respectively, through a hole in the zona pellucida using Piezo-pulse drilling (Primetech, Ibaraki, Japan, http://www.primetech.co.jp) , and all of the blastomeres were separated by repeated pipetting.1 R4 f/ m2 d0 C4 ~
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Figure 1. This diagram illustrates the production of ES/parthenogenetic ES cell lines from single blastomeres or polar bodies. Experiment 1: Blastomeres from two-cell, early four-cell, late four-cell, and eight-cell stage embryos were collected at 40, 48, 62, and 64 hours after human chorionic gonadotropin injection, respectively, with a micromanipulator. These blastomeres were immediately plated onto ES cell culture medium with MEFs. Experiment 2: First polar bodies were taken from oocytes and injected into enucleated oocytes. The reconstructed oocytes were activated with cytochalasin B to prevent extrusion of the second polar body. Second polar bodies were taken from zygotes and fused with preactivated (3 hours before use) enucleated oocytes. The next day, when these reconstructed embryos had developed to the two-cell stage, both blastomeres were fused to each other to produce diploid nuclei. When these embryos reached the blastocyst stage after 4 days in culture, the zonae pellucidae were removed, and the embryos were plated onto ES cell culture medium with MEFs. In both experiments, if some of the plated cells developed and formed a clump approximately 2 weeks later, they were passaged in the usual manner. Abbreviations: 2PB, second polar body; ES, embryonic stem; MEF, mouse embryonic feeder cell.

Establishment of ES Cells from Polar Bodies

Nuclear transfer of the first polar bodies .8 [! I# [# ]$ H2 q. x2 I5 }2 w3 ?

The second polar bodies derived from B6D2F2 zygotes were drawn into the pipette and inserted through the zona pellucida into the perivitelline space of B6D2F1 preactivated enucleated oocytes. These were then transferred to a fusion chamber with electrodes separated by 0.5 mm with calcium-free fusion medium (0.3 M mannitol, 0.1 mM MgSO4, and 0.1% polyvinyl alcohol). A pulse of 20 V at 1 MHz alternating current was applied for 2 seconds to align the oocyte and the second polar body. Immediately after this, 100 V of direct current was applied for 20 milliseconds to induce fusion. The oocyte-polar body mosaics were kept for 5 minutes in the chamber, and then transferred to CZB medium  and incubated at 37¡ãC under 5% CO2 in air for 18 hours. When those reconstructed embryos developed to the two-cell stage, they were again transferred to a fusion chamber and fused with each other using the same protocol to produce diploid parthenogenetic embryos.1 |3 O9 G7 F. e  A
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Polymerase Chain Reaction Analysis of Genomic DNA2 _: P% Z4 m* Q8 z- Y
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To exclude the possibility that the established pES cell lines were derived from intact F1 oocytes rather than from polar bodies, the first polar bodies were taken from the 129/Sv mouse strain, which can be distinguished from the oocyte donor by its genomic DNA. The microsatellite markers D1Mit26, D3Mit18, and D3Mit21 were amplified using primer pair sequences obtained from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). DNA was extracted from ear biopsy samples and pES cells. Thirty cycles of polymerase chain reaction (PCR) were performed, and the products were separated on 3% agarose gel before visualization.! Q6 Z6 w1 k5 h& U

Reverse Transcription PCR Analysis of Oct3/4 and Nanog Expression
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Reverse transcriptase (RT)-PCRs to detect Oct3/4 and Nanog transcripts were performed basically following a previously reported method , with slight changes in cDNA preparation and the use of Taq DNA polymerase. Total RNA from ES, pES, and fibroblast cells was prepared using the RNeasy Protect Mini Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) and was reverse transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen). Ex Taq Hot Start Version (Takara, Shiga, Japan, http://www.takara.co.jp) was used in the PCR. The primers used for amplification of each DMRs were Oct3/4-F, 5'-CCTGCAGAAGGAGCTAGAACAGT-3'; Oct3/4-R, 5'-TGTTCTTAAGGCTGAGCTGCAA-3'; Nanog-F, 5'-TGTGTGCACTCAAGGACAGGTT-3'; Nanog-R, 5'-TCAGGTTCAGAATGGAGGAGAGTT-3'.
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Karyotype Analysis, Immunofluorescence, and Alkaline Phosphatase Staining
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All established ES cell lines were tested for pluripotency by alkaline phosphatase staining for the primordial germ-cell phenotype, according to the manufacturer's protocol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Twenty-four randomly selected cell lines (four cell lines from each experiment) were also examined in more detail using the ES-cell-specific markers Oct3/4, Nanog, and stage-specific embryonic antigen (SSEA)-1, and markers negative for mouse ES cells, SSEA-3 and SSEA-4. Immunohistochemistry was performed using the following monoclonal antibodies: anti-Oct3/4 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com); anti-Nanog (1:200; ReproCELL, Tokyo, http://www.reprocell.com/en); anti-SSEA-1, 3, (1:100; Chemicon, Temecula, CA, http://www.chemicon.com), anti-SSEA-4 (1:100; Santa Cruz Biotechnology). Alexa Fluor 488-, 350-, or 568-labeled secondary antibodies (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) were used for detection as appropriate. The cell karyotypes were also examined using Giemsa staining and chromosome painting with spectral karyotyping fluorescent in situ hybridization (SKY-FISH; Applied Spectral Imaging, Carlsbad, CA, http://www.spectral-imaging.com), according to the manufacturer's instructions .6 J" O" W+ t/ u
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Production of Chimeric Mice and Confirmation of Germline Transmission of ES and pES Cells/ p8 m6 `: |; ?: C

Two of each ES or pES cell line derived from all stage embryos, with or without GFP, were randomly selected, and those cells were introduced into the blastocoels of (albino) E3.5 ICR strain blastocysts by Piezo-assisted microinjection (Primetech) to produce chimeric embryos. Immediately after injection, the blastocysts were transferred into pseudopregnant ICR-strain surrogate mothers . The chimeric mice derived from the F2 blastomere ES cells had black eyes and gray or dark-colored coats, whereas those from the GFP-ES cell chimeric mice could be distinguished by their green color under ultraviolet (UV) light. The chimeric mice derived from 129/Sv polar body pES cells had an agouti coat color. When mature, the chimeric offspring with colored coats were selected at random and mated with ICR strain mice to examine the germline transmission. In some case, the chimeric mice constructed using ES cells derived from transgenic mice expressing GFP were killed and dissected, and any GFP expression in whole tissues or sections was detected using a UV lamp.

Production of Offspring from Biopsied Embryos; S/ D+ y8 x$ A( ^4 X& u2 R, c

In some experiments, the biopsied or intact control embryos were transferred to the oviducts of 0.5 days postcoitus (dpc) pseudopregnant mothers to examine the effects of the biopsy treatment on full-term development. The number of offspring was examined at 19.5 dpc.
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Statistical Analysis
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Outcomes were evaluated using 2 tests, and p

RESULTS" U; P6 \5 U) E5 K

Embryo Stage Is Important in the Establishment of ES Cell Lines# @$ l! T/ B& K3 `! B; K
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In the first set of experiments, we tested which stage of the embryos could be used to establish ES cell lines from single blastomeres (Table 1). All stages of blastomeres developed to blastocysts in 3¨C5 days in ES cell establishment medium (Fig. 2A¨C2C). Interestingly, we found that ES cell lines could be established easily from all stages of blastomeres without the use of other ES cells as supporting cells. The establishment rates declined with increasing embryo age, from 69% at the two-cell stage to 14% at the eight-cell stage in F2 embryos. Importantly, the rates were significantly different between the early (40%) and late four-cell stage embryos (22%), even though these blastomeres have the same cytoplasmic volume (Table 1). In the control, 88% of whole BDF2 blastocysts formed ES cell lines. The same tendency was observed when GFP-expressing embryos were used.& K" X1 o3 P& X5 F

Figure 2. Pluripotency of ES cells derived from single blastomeres. (A¨CC): All blastomeres formed blastocysts in ES cell culture medium. (A): Two-cell blastomere 5 days after culture. (B): Four-cell blastomere at 3 days. (C): Eight-cell blastomere at 3 days. (D): Phase contrast image of pES cell derived from a first polar body (line pES1¨C1). (E¨CG): ES cells derived from an eight-cell blastomere (line F2¨C1/8¨C1). Cells positive for the expression of the pluripotency markers Oct3/4 (E), Nanog (F), and alkaline phosphatase (G). (H): Spectral karyotyping fluorescent in situ hybridization painting shows the normal karyotype of the ES cell. (I): ES cells contributed to all the tissues of chimeric mice, including the germ line. (J): Polymerase chain reaction (PCR) analyses of microsatellite markers demonstrate that the pES cell lines were derived from the first polar bodies of the 129/Sv mouse strain. (K): reverse transcriptase (RT)-PCR analyses of ES and pES cell lines, with the usual ES cells (C57BL/6) and mouse embryonic feeder cells as the positive and negative controls, respectively. The gene expression patterns of Oct3/4 and Nanog analyzed by RT-PCR were consistent with the staining results when the corresponding antibodies were used. Abbreviation: BD, B6D2F1 mouse; ES, embryonic stem; M, marker; Neg, negative; PB, polar body; pES, parthenogenetic ES; Posi, positive.2 |2 t7 M$ X# o0 I) u9 x

Table 1. Effect of the developmental stage of the embryo on embryonic stem cell establishment from blastomeres8 z" t5 m: t0 z8 Q

Polar Bodies Can Be Used to Establish pES Cell Lines% y  P( N8 Q  ~( ^. A8 u

In this experiment, we tested whether first and second polar bodies can be used as sources for the establishment of pES cells. As shown in Table 2, although the rate of blastocyst development of reconstructed embryos was not as high as that of the control parthenogenetic embryos (21%¨C63% vs. 100%, respectively), interestingly, once the embryos had developed into blastocysts, these polar-body-derived embryos could be used relatively easily to establish pES cell lines (36%¨C40% for the first polar bodies and 33% for the second polar bodies, which are similar to the rate of 42% for the control whole parthenogenetic embryos). PCR analyses of microsatellite markers in genomic DNA from the ears of the BDF1 and 129/Sv mice and from the pES cell lines confirmed their genetic identity. The polymorphic markers D1Mit26, D3Mit18, and D3Mit21 were present in the genomic DNA of the 129/Sv mice and that from the pES cell lines, but differed from the markers in the oocyte recipient strain B6D2F1 (Fig. 2J)., n$ \1 X% G/ B
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Table 2. Effect of the development stage of the embryo on embryonic stem cell establishment from blastomeres( h( e6 r( c3 P4 d

Developmental Potential of Embryos After Removal of a Single Blastomere

The biopsied embryos survived well after transfer into pseudopregnant recipients. Of the eight-cell embryos transferred, 22 (47%) of 47 embryos gave rise to live young; of the four-cell embryos, 4 (67%) of 6 embryos gave rise to live young, which is similar to the rate for intact embryos . Although more experiments are required, this suggests that the early four-cell embryo stage is optimal for both the establishment of ES cell lines (40%) and the production of live offspring.; X8 g9 m3 ]# U* b) W

Examination of Each Blastomere of Individual Embryos for ES Cell Establishment
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To determine the potential of each blastomere of the same embryo to form ES cells, we tried to use all blastomeres from individual embryos to establish ES cell lines. In this series of experiments, the zona pellucida was removed with acid Tyrode's solution, and the naked two-cell, late four-cell, and eight-cell embryos were pipetted several times until the blastomeres were successfully separated. We also tried to use early four-cell embryos but were unable to disperse all the blastomeres from any single embryo. In this experiment, we established two ES cell lines, both from the blastomeres of only one two-cell embryo (Table 3). However, this at least demonstrates that both these blastomeres had equal potential. In contrast, no ES cell lines were established from all the blastomeres of the same embryo at the four-cell or eight-cell stage. The total establishment rates (33%, 8%, and 8% for the two-cell, late four-cell, and eight-cell embryos, respectively) were lower than those in the first experiment (Table 1). In this procedure, the zona pellucida was removed with acid treatment, and the naked embryos were pipetted several times to separate the blastomeres. This may have damaged the cells so that they could not develop in ES medium . Further experiments, including the surgical removal of each blastomere, are required.
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Table 3. Establishment rates for ES cell lines from all blastomeres of individual embryos- A3 _$ O0 S' Q9 N/ @  S* P2 C

Table 4. Generation of chimeric mice and germ line transmission of embryonic stem cells3 e# k7 l* S1 J
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Examination of ES-Cell-Specific Marker Expression in ES/pES Cells
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The newly established ES cells were analyzed for pluripotency by immunostaining for alkaline phosphatase, Oct3/4, Nanog, and SSEA-1, and were examined for embryoid body formation. We examined the expression of SSEA-3 and -4 in the ES cells as negative controls. All 24 randomly selected ES cell lines examined were positive for the ES-cell-specific markers Oct3/4 and Nanog (Fig. 2D¨C2F), and were negative for the negative control markers, in a pattern similar to that observed in normally fertilized ES cells. We gave up using SSEA-1 as a positive marker because even the control ES cells showed no clear positive signal in our laboratory. These immunohistochemical data for ES and pES cells were also confirmed by RT-PCR analysis (Fig. 2K). The gene expression patterns for Oct3/4 and Nanog were consistent with the staining results obtained with their respective specific antibodies. We found no differences in the levels of the expression of these genes. For karyotype analysis, approximately 80% of the cells from each cell line (two derived from each stage embryo and two from first polar bodies) had normal karyotypes, as shown by Giemsa staining and SKY-FISH chromosome painting (Fig. 2H). These data are within the normal range (35%¨C73%) for normal ES and nuclear transfer-ES cell lines .

Contribution of ES Cells to Chimeric Mice
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To evaluate the possible pluripotency of these ES cells, chimeric mice were produced by injecting the ES cells or pES cells into the blastocoels of ICR-strain blastocysts, which were then transferred into pseudopregnant recipients. Evidence of the coat color contribution made by the ES cells (dark, brown, or gray for F2 ES cells; agouti for 129/Sv pES cells) was identified by the detection of the donor ES cell coat colors of the chimeras. ES chimeras with a strong coat color contribution (more than half the coat color) were obtained from all the cell lines examined, except line F2¨C1/2, which was derived from a two-cell stage embryo. After sexual maturation, these chimeric mice were mated with ICR strain mice. So far, at least one chimeric mouse derived from a blastomere of each stage embryo has delivered nonalbino offspring or offspring that express the gene for GFP, demonstrating true germline transmission of the ES cell genotype (Fig. 2I). The pES cell lines derived from polar bodies were established recently, and mating experiments are still proceeding. The chimerism varied between cell lines, especially for pES cells, which have shown lower coat-color contributions than other cells. However, because the contribution rate of ES cells to chimeric mice is affected not only by the quality of the cell lines but also by the conditions of each experiment, it is unclear which cell line in this experiment was most highly pluripotent. Furthermore, when the organs of the dark-coat-colored chimeric mice derived from GFP-expressing ES cells (line GFP-1/2) were examined, all of the tissues examined (brain, lung, heart, stomach, thymus, kidney, spleen, intestines, liver, and pancreas) exhibited green GFP emission under UV illumination, in either whole tissues or sections (data not shown).

DISCUSSION
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The main objective of this study was to evaluate the possibility of producing ES cell lines from single blastomeres obtained by biopsy from different stage mouse embryos. We have demonstrated that all stages of embryos can be used to establish ES cell lines with relatively high success rates (14%¨C69%) without the use of other ES cells as supporting cells. The biopsied embryos developed to full term without a significant reduction in survival, irrespective of embryo stage. Polar bodies from oocytes or zygotes could also be used to establish pES cell lines with success rates of 33%¨C40%, and those cell lines could not be distinguished either morphologically or by gene expression.; e3 h) L( h+ E- t, g# c5 e4 g
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In 1996, Delhaise et al. . The isolated blastomeres were each aggregated with a small clump of previously established GFP-expressing ES cells, plated onto MEFs, and cultured in ES cell growth medium. After the cells had proliferated, the GFP-negative cells were separated from the GFP-positive ES cells. The establishment rate from single blastomeres was low in both mice and humans (4% and 2%, respectively). The authors suggested that such ES cell coculture was critical to the success of this system, but it was unclear whether this was attributable to substances secreted by the ES cells or a requirement for cell-cell contact.
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In contrast, we have demonstrated that ES cell lines can be established easily from single blastomeres of all stages of embryos with high success rates, using a simple method of culture on MEFs. We did not use aggregation with other ES cells, so this success rate is probably attributable to the new ES cell medium  reported that when 5¨C50 µM ACTH was added to the medium, single ES cells could be propagated without losing their pluripotency. It seems that this medium can support single blastomere development, even from two- to eight-cell stage embryos to the establishment of blastocysts, and also ES cells, more efficiently than can the method based on aggregation with other ES cells.$ c7 \( A5 I6 j* f

One of the more interesting results we obtained occurred when we compared early and late four-cell stage embryos. The establishment rate of ES cell lines decreased significantly from early to late stage (40% vs. 22%, respectively). This was despite these blastomeres' having the same cytoplasmic volumes (Table 1). We inferred that the first differentiation occurs between the early and late four-cell stages, and, thus, that some of the blastomeres had lost pluripotency at the late four-cell stage. Although it has previously been thought that the first differentiation occurs in mouse embryogenesis at the 8¨C16-cell stage . We hope that our data may provide additional results to this discussion." L2 Y- g$ y! d

This is the first report to demonstrate that the first and second polar bodies can be used as a source in the generation of parthenogenetic ES cells. These are not altogether surprising results insofar as previous studies have already demonstrated that both first and second polar bodies can participate as female pronuclei in full-term development when reconstructed oocytes are fertilized with spermatozoa  suggest that parthenogenetic embryos do not develop to term because of a high frequency of errors in the X-chromosome inactivation that occurs in extraembryonic tissues when both X chromosomes are derived only from the female. One might conclude that imprinting has a significant effect on extraembryonic tissues but not on the inner cell mass from which our stem cells were derived.2 R. B3 K, f7 A7 f- r; R0 A* [4 s
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In conclusion, our results suggest that the single cells of all early-stage embryos or polar bodies can potentially be converted into ES cells without any special treatment. Although this technique may allow ES cell banking of autologous ES cell lines by increasing the numbers of possible lines, it is difficult to establish a baby's own ES cell line at birth with any certainty. Here, we have demonstrated that, under optimal conditions, the rate of establishing ES cells from biopsied embryos can be up to 40% and the biopsied embryos can develop to full term at normal rates. For humans, this suggests that embryo biopsy for preimplantation genetic diagnosis may offer the additional benefit of allowing autologous ES cell lines to be laid down.! o) ^3 t# c4 j$ k/ ?0 z; O1 {
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST: z* b; [# x( |$ B; J# S
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The authors indicate no potential conflicts of interest.+ N  U" p2 A+ ~5 A& i
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