- Email: [email protected]
Establishment of mouse embryonic stem cell lines from somatic cell nuclei by nuclear transfer into aged, fertilization-failure mouse oocytes
Current Biology Vol 17 No 4 R120
Correspondences
Establishment of mouse embryonic stem cell lines from somatic cell nuclei by nuclear transfer into aged, fertilization-failure mouse oocytes Sayaka Wakayama, Rinako Suetsugu, Nguyen Van Thuan, Hiroshi Ohta, Satoshi Kishigami and Teruhiko Wakayama* It has been widely assumed that fresh oocytes are required for the establishment of embryonic stem cells (ES cells) via somatic cell nuclear transfer (SCNT). Such cell lines are referred to as ‘NT-ES cells’ [1]. This presumed requirement, however, gives rise to fundamental ethical concerns surrounding its potential application to human cells, as fresh oocytes must be obtained from a healthy female donor. Human in vitro fertilization (IVF) is now performed routinely in infertility clinics, but in the IVF attempts some of the oocytes fail to become fertilized and are discarded [2]. Here we show that such aged fertilization-failure (AFF) oocytes from mice can be successfully used as recipients in nuclear transfer. We suggest that, if recapitulated with human oocytes, this method may provide a means of avoiding some of the ethical concerns surrounding fresh oocyte donation for nuclear transfer. AFF oocytes lacking a second polar body and pronucleus (see Supplemental data published with this article online) were selected at six hours after insemination to serve as recipients for nuclear transfer. The somatic donor cells (cumulus cells) were collected from several mouse strains, including BDF1 — a strain engineered to express green
fluorescent protein (GFP) in all body cells. Nuclear transfer and NT-ES cell establishment were performed as described [3,4], with some modifications to timing (Supplemental data). The success rate of nuclear transfer and development to morula or blastocyst stages was significantly lower for AFF oocytes than for fresh oocytes (Figure 1; Table 1). At the two-cell stage, embryos derived from AFF oocytes exhibited small fragments between blastomeres (Figure 1B), which disappeared by the 4- or 8-cell stage. However, the rate of establishment of NT-ES cell lines from cloned morulae or blastocyststage embryos was similar for AFF oocytes and fresh oocytes (6% vs. 7%; Table 1; Supplemental data). Surprisingly, even when oocytes from several mouse strains were stored at room temperature for one day (24 h after oocyte collection), they could be used to establish NTES cell lines at similar efficiencies (4%; Table 1; Supplemental data). It is known that oocytes stored at low temperature suffer irreversible damage to spindle structures [5,6]. All established cell lines stained positive for the ES cell-specific markers alkaline phosphatase, Figure 1. NT-ES cell lines established from AFF mouse oocytes. (A) Success rate from recon structed oocyte to NT-ES cell establishment. AFF oocytes were weaker for manipulation and in vitro development, but the rate of establishment of NT-ES cell lines was similar to that achieved using fresh oocytes. (‘Survive’: survival after nuclear injection; 'PN': pseudo-pronuclear formation after activation; ‘Mor/Blast’: Morulae or blastocyst develo pment at 4 days after NT.) (B) Two-cell stage cloned embryos derived from AFF oocytes; almost all embryos show small fragments between blastomeres (arrows). (C) Two-cell stage cloned embryos derived from fresh oocytes. (D,E) Tetraploid complementation chimeric mouse. Expression of GFP suggests that the NT-ES cells contribute to all functional organs, except the placenta (E).
Oct3/4 and Nanog, and all randomly selected NT-ES cell lines (n = 13) were of normal karyotype (Supplemental Data). To demonstrate the pluripotency of these NT-ES cell lines, BDF1-GFP derived NT-ES cells were injected into the perivitelline space of either diploid or tetraploid mouse embryos. We obtained a number of healthy and fertile offspring using both methods (Figure 1D; Supplemental data). GFP expression and coat color confirmed that these NT-ES cell lines are truly derived from donor nuclei. Analyses of chimeric contribution showed that these cells are capable of functional differentiation into all cell types required for development and germ cell formation, similar to ES and NT-ES cells derived from fresh oocytes [7]. We additionally examined the full-term developmental potential of cloned embryos and of embryos produced by intracytoplasmic sperm injection (ICSI) using AFF oocytes. After ICSI, AFF oocytes showed much lower rates of development to term than fresh oocytes. In the NT experiments, none of the cloned embryos developed to term when
A 100 Fresh Failed IVF
80 60 %
40 20 0 Survive
Mor/Blast Establish
PN
B
C
D
E
Current Biology
Magazine R121
Table 1. Establishment of NT-ES cell lines, production of cloned and ICSI offspring using AFF oocytes. Type of experiment
Type of oocyte
Number of oocytes used
Embryos developed to blastocyst stage (%)
Number of established cell lines (%)
Embryos developed to 2-cell stage (%)
Number of offspring (%)
NT-ES
AFF
430
112 (26)
27 (6)
-
-
24h stored
229
35 (15)
10 (4)
-
-
Fresh
162
83 (51)
12 (7)
-
-
AFF
797
-
-
357 (45)
0
Fresh
162
-
-
119 (73)
3 (1.9)
AFF
60
-
-
36 (60)
4 (6.7)
Fresh
37
-
-
32 (86)
25 (68)
Clone
ICSI
AFF oocytes were used (Table 1; Supplemental data). In general, the success rate of producing full-term offspring from cloned embryos is very low compared to that from ICSI embryos [3,6]. It thus appears to be more difficult to produce cloned mice from AFF oocytes. This may be due to a partial reduction or alteration in the genomic reprogramming potential of such oocytes that leaves them sufficient for the generation of mouse NT-ES cells but insufficient to support fullterm development. The idea of using AFF oocytes instead of fresh oocytes has already been attempted in human NT experiments [8–10]. However, human AFF oocytes have been shown to exhibit multiple abnormalities, such as aberrant spindles and transcriptional profiles [10]. Therefore, previous reports have concluded that AFF oocytes are relatively inefficient recipients for use in human NT. However, although aged mouse oocytes have also been shown to bear similar abnormalities, most of such aberrations are believed to occur in the spindle, rather than the cytoplasm, as the developmental arrest could not be rescued when spindles from aged oocytes were transferred into fresh oocytes [6]. It seems likely that mouse oocyte cytoplasm is more tolerant of aging than human oocytes. However, in human NT experiments, even when fresh oocytes were used, embryonic development was still limited, suggesting that technical details
of the nuclear transfer protocol are still imperfect and in need of optimization for use in human NT. Previously, we believed that fresh oocytes were essential for mouse NT experiments. However, after improvements to the mouse NT protocol [11], we clearly demonstrate that AFF oocytes can be used to generate mouse NT-ES cells. Future improvements may make this technqiue applicable for the derivation of human NT-ES cells as well. Acknowledgments We thank Dr. Jim M. Cummins and Mr. D. Sipp for critical and useful comments on the manuscript. We are grateful to the Laboratory for Animal Resources and Genetic Engineering for the housing of mice. This work was supported by a Scientific Research in Priority Areas (15080211) and a project for the realization of regenerative medicine (research toward technical developments in stem cell manipulation) to T.W. from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Supplemental data Supplemental data including experimental procedures, tables and figures can be found at http://www.current-biology.com/cgi/content/full/17/4/R120/DC1 References 1. Wakayama, T., Tabar, V., Rodriguez, I., Perry, A.C., Studer, L., and Mombaerts, P. (2001). Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 292, 740–743. 2. Nagy, Z.P., Joris, H., Liu, J., Staessen, C., Devroey, P., and Van Steirteghem, A.C. (1993). Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes. Hum. Reprod. 8, 2180–2184.
3. Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R., and Yanagimachi, R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374. 4. Kishigami, S., Wakayama, S., Thuan, N.V., Ohta, H., Mizutani, E., Hikichi, T., Bui, H.T., Balbach, S., Ogura, A., Boiani, M., et al. (2006). Production of cloned mice by somatic cell nuclear transfer. Nat. Prot. 1, 125–138. 5. Pickering, S.J., Braude, P.R., Johnson, M.H., Cant, A., and Currie, J. (1990). Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil. Steril. 54, 102–108. 6. Wakayama, S., Thuan, N.V., Kishigami, S., Ohta, H., Mizutani, E., Hikichi, T., Miyake, M., and Wakayama, T. (2004). Production of offspring from one-day-old oocytes stored at room temperature. J. Reprod. Dev. 50, 627–637. 7. Wakayama, S., Jakt, M.L., Suzuki, M., Araki, R., Hikichi, T., Kishigami, S., Ohta, H., Van Thuan, N., Mizutani, E., Sakaide, Y., et al. (2006). Equivalency of nuclear transfer-derived embryonic stem cells to those derived from fertilized mouse blastocysts. Stem Cells 24, 2023–2033. 8. Stojkovic, M., Stojkovic, P., Leary, C., Hall, V.J., Armstrong, L., Herbert, M., Nesbitt, M., Lako, M., and Murdoch, A. (2005). Derivation of a human blastocyst after heterologous nuclear transfer to donated oocytes. Reprod. Biomed. Online 11, 226–231. 9. Lavoir, M.C., Weier, J., Conaghan, J., and Pedersen, R.A. (2005). Poor development of human nuclear transfer embryos using failed fertilized oocytes. Reprod. Biomed. Online 11, 740–744. 10. Hall, V.J., Compton, D., Stojkovic, P., Nesbitt, M., Herbert, M., Murdoch, A., and Stojkovic, M. (2007). Developmental competence of human in vitro aged oocytes as host cells for nuclear transfer. Hum. Reprod. 22, 52–62. 11. Kishigami, S., Mizutani, E., Ohta, H., Hikichi, T., Thuan, N.V., Wakayama, S., Bui, H.T., and Wakayama, T. (2006). Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer. Biochem. Biophys. Res. Commun. 340, 183–189.
Center for Developmental Biology, RIKEN Kobe, 2-2-3 Minatojimaminamimachi, Kobe 650-0047, Japan. *E-mail: [email protected]
Correspondences
Establishment of mouse embryonic stem cell lines from somatic cell nuclei by nuclear transfer into aged, fertilization-failure mouse oocytes Sayaka Wakayama, Rinako Suetsugu, Nguyen Van Thuan, Hiroshi Ohta, Satoshi Kishigami and Teruhiko Wakayama* It has been widely assumed that fresh oocytes are required for the establishment of embryonic stem cells (ES cells) via somatic cell nuclear transfer (SCNT). Such cell lines are referred to as ‘NT-ES cells’ [1]. This presumed requirement, however, gives rise to fundamental ethical concerns surrounding its potential application to human cells, as fresh oocytes must be obtained from a healthy female donor. Human in vitro fertilization (IVF) is now performed routinely in infertility clinics, but in the IVF attempts some of the oocytes fail to become fertilized and are discarded [2]. Here we show that such aged fertilization-failure (AFF) oocytes from mice can be successfully used as recipients in nuclear transfer. We suggest that, if recapitulated with human oocytes, this method may provide a means of avoiding some of the ethical concerns surrounding fresh oocyte donation for nuclear transfer. AFF oocytes lacking a second polar body and pronucleus (see Supplemental data published with this article online) were selected at six hours after insemination to serve as recipients for nuclear transfer. The somatic donor cells (cumulus cells) were collected from several mouse strains, including BDF1 — a strain engineered to express green
fluorescent protein (GFP) in all body cells. Nuclear transfer and NT-ES cell establishment were performed as described [3,4], with some modifications to timing (Supplemental data). The success rate of nuclear transfer and development to morula or blastocyst stages was significantly lower for AFF oocytes than for fresh oocytes (Figure 1; Table 1). At the two-cell stage, embryos derived from AFF oocytes exhibited small fragments between blastomeres (Figure 1B), which disappeared by the 4- or 8-cell stage. However, the rate of establishment of NT-ES cell lines from cloned morulae or blastocyststage embryos was similar for AFF oocytes and fresh oocytes (6% vs. 7%; Table 1; Supplemental data). Surprisingly, even when oocytes from several mouse strains were stored at room temperature for one day (24 h after oocyte collection), they could be used to establish NTES cell lines at similar efficiencies (4%; Table 1; Supplemental data). It is known that oocytes stored at low temperature suffer irreversible damage to spindle structures [5,6]. All established cell lines stained positive for the ES cell-specific markers alkaline phosphatase, Figure 1. NT-ES cell lines established from AFF mouse oocytes. (A) Success rate from recon structed oocyte to NT-ES cell establishment. AFF oocytes were weaker for manipulation and in vitro development, but the rate of establishment of NT-ES cell lines was similar to that achieved using fresh oocytes. (‘Survive’: survival after nuclear injection; 'PN': pseudo-pronuclear formation after activation; ‘Mor/Blast’: Morulae or blastocyst develo pment at 4 days after NT.) (B) Two-cell stage cloned embryos derived from AFF oocytes; almost all embryos show small fragments between blastomeres (arrows). (C) Two-cell stage cloned embryos derived from fresh oocytes. (D,E) Tetraploid complementation chimeric mouse. Expression of GFP suggests that the NT-ES cells contribute to all functional organs, except the placenta (E).
Oct3/4 and Nanog, and all randomly selected NT-ES cell lines (n = 13) were of normal karyotype (Supplemental Data). To demonstrate the pluripotency of these NT-ES cell lines, BDF1-GFP derived NT-ES cells were injected into the perivitelline space of either diploid or tetraploid mouse embryos. We obtained a number of healthy and fertile offspring using both methods (Figure 1D; Supplemental data). GFP expression and coat color confirmed that these NT-ES cell lines are truly derived from donor nuclei. Analyses of chimeric contribution showed that these cells are capable of functional differentiation into all cell types required for development and germ cell formation, similar to ES and NT-ES cells derived from fresh oocytes [7]. We additionally examined the full-term developmental potential of cloned embryos and of embryos produced by intracytoplasmic sperm injection (ICSI) using AFF oocytes. After ICSI, AFF oocytes showed much lower rates of development to term than fresh oocytes. In the NT experiments, none of the cloned embryos developed to term when
A 100 Fresh Failed IVF
80 60 %
40 20 0 Survive
Mor/Blast Establish
PN
B
C
D
E
Current Biology
Magazine R121
Table 1. Establishment of NT-ES cell lines, production of cloned and ICSI offspring using AFF oocytes. Type of experiment
Type of oocyte
Number of oocytes used
Embryos developed to blastocyst stage (%)
Number of established cell lines (%)
Embryos developed to 2-cell stage (%)
Number of offspring (%)
NT-ES
AFF
430
112 (26)
27 (6)
-
-
24h stored
229
35 (15)
10 (4)
-
-
Fresh
162
83 (51)
12 (7)
-
-
AFF
797
-
-
357 (45)
0
Fresh
162
-
-
119 (73)
3 (1.9)
AFF
60
-
-
36 (60)
4 (6.7)
Fresh
37
-
-
32 (86)
25 (68)
Clone
ICSI
AFF oocytes were used (Table 1; Supplemental data). In general, the success rate of producing full-term offspring from cloned embryos is very low compared to that from ICSI embryos [3,6]. It thus appears to be more difficult to produce cloned mice from AFF oocytes. This may be due to a partial reduction or alteration in the genomic reprogramming potential of such oocytes that leaves them sufficient for the generation of mouse NT-ES cells but insufficient to support fullterm development. The idea of using AFF oocytes instead of fresh oocytes has already been attempted in human NT experiments [8–10]. However, human AFF oocytes have been shown to exhibit multiple abnormalities, such as aberrant spindles and transcriptional profiles [10]. Therefore, previous reports have concluded that AFF oocytes are relatively inefficient recipients for use in human NT. However, although aged mouse oocytes have also been shown to bear similar abnormalities, most of such aberrations are believed to occur in the spindle, rather than the cytoplasm, as the developmental arrest could not be rescued when spindles from aged oocytes were transferred into fresh oocytes [6]. It seems likely that mouse oocyte cytoplasm is more tolerant of aging than human oocytes. However, in human NT experiments, even when fresh oocytes were used, embryonic development was still limited, suggesting that technical details
of the nuclear transfer protocol are still imperfect and in need of optimization for use in human NT. Previously, we believed that fresh oocytes were essential for mouse NT experiments. However, after improvements to the mouse NT protocol [11], we clearly demonstrate that AFF oocytes can be used to generate mouse NT-ES cells. Future improvements may make this technqiue applicable for the derivation of human NT-ES cells as well. Acknowledgments We thank Dr. Jim M. Cummins and Mr. D. Sipp for critical and useful comments on the manuscript. We are grateful to the Laboratory for Animal Resources and Genetic Engineering for the housing of mice. This work was supported by a Scientific Research in Priority Areas (15080211) and a project for the realization of regenerative medicine (research toward technical developments in stem cell manipulation) to T.W. from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Supplemental data Supplemental data including experimental procedures, tables and figures can be found at http://www.current-biology.com/cgi/content/full/17/4/R120/DC1 References 1. Wakayama, T., Tabar, V., Rodriguez, I., Perry, A.C., Studer, L., and Mombaerts, P. (2001). Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 292, 740–743. 2. Nagy, Z.P., Joris, H., Liu, J., Staessen, C., Devroey, P., and Van Steirteghem, A.C. (1993). Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes. Hum. Reprod. 8, 2180–2184.
3. Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R., and Yanagimachi, R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374. 4. Kishigami, S., Wakayama, S., Thuan, N.V., Ohta, H., Mizutani, E., Hikichi, T., Bui, H.T., Balbach, S., Ogura, A., Boiani, M., et al. (2006). Production of cloned mice by somatic cell nuclear transfer. Nat. Prot. 1, 125–138. 5. Pickering, S.J., Braude, P.R., Johnson, M.H., Cant, A., and Currie, J. (1990). Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil. Steril. 54, 102–108. 6. Wakayama, S., Thuan, N.V., Kishigami, S., Ohta, H., Mizutani, E., Hikichi, T., Miyake, M., and Wakayama, T. (2004). Production of offspring from one-day-old oocytes stored at room temperature. J. Reprod. Dev. 50, 627–637. 7. Wakayama, S., Jakt, M.L., Suzuki, M., Araki, R., Hikichi, T., Kishigami, S., Ohta, H., Van Thuan, N., Mizutani, E., Sakaide, Y., et al. (2006). Equivalency of nuclear transfer-derived embryonic stem cells to those derived from fertilized mouse blastocysts. Stem Cells 24, 2023–2033. 8. Stojkovic, M., Stojkovic, P., Leary, C., Hall, V.J., Armstrong, L., Herbert, M., Nesbitt, M., Lako, M., and Murdoch, A. (2005). Derivation of a human blastocyst after heterologous nuclear transfer to donated oocytes. Reprod. Biomed. Online 11, 226–231. 9. Lavoir, M.C., Weier, J., Conaghan, J., and Pedersen, R.A. (2005). Poor development of human nuclear transfer embryos using failed fertilized oocytes. Reprod. Biomed. Online 11, 740–744. 10. Hall, V.J., Compton, D., Stojkovic, P., Nesbitt, M., Herbert, M., Murdoch, A., and Stojkovic, M. (2007). Developmental competence of human in vitro aged oocytes as host cells for nuclear transfer. Hum. Reprod. 22, 52–62. 11. Kishigami, S., Mizutani, E., Ohta, H., Hikichi, T., Thuan, N.V., Wakayama, S., Bui, H.T., and Wakayama, T. (2006). Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer. Biochem. Biophys. Res. Commun. 340, 183–189.
Center for Developmental Biology, RIKEN Kobe, 2-2-3 Minatojimaminamimachi, Kobe 650-0047, Japan. *E-mail: [email protected]