Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos

Eunju Kang, Guangming Wu, Hong Ma, Ying Li, Rebecca Tippner-Hedges, Masahito Tachibana, Michelle Sparman, Don P. Wolf, Hans R. Schöler, Shoukhrat Mitalipov

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative applications, as donated or discarded embryos are more accessible than unfertilized MII oocytes.

Original languageEnglish (US)
Pages (from-to)101-104
Number of pages4
JournalNature
Volume508
Issue number7498
DOIs
StatePublished - 2014

Fingerprint

Interphase
Cytoplasm
Embryonic Structures
Metaphase
Embryonic Stem Cells
Oocytes
Zygote
Embryo Transfer
Cell Nucleus
Cell Cycle
Cumulus Cells
Cellular Reprogramming
Tetraploidy
Blastocyst
Fertilization
Organism Cloning
Fibroblasts
Mothers

ASJC Scopus subject areas

  • General

Cite this

Kang, E., Wu, G., Ma, H., Li, Y., Tippner-Hedges, R., Tachibana, M., ... Mitalipov, S. (2014). Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos. Nature, 508(7498), 101-104. https://doi.org/10.1038/nature13134

Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos. / Kang, Eunju; Wu, Guangming; Ma, Hong; Li, Ying; Tippner-Hedges, Rebecca; Tachibana, Masahito; Sparman, Michelle; Wolf, Don P.; Schöler, Hans R.; Mitalipov, Shoukhrat.

In: Nature, Vol. 508, No. 7498, 2014, p. 101-104.

Research output: Contribution to journalArticle

Kang, E, Wu, G, Ma, H, Li, Y, Tippner-Hedges, R, Tachibana, M, Sparman, M, Wolf, DP, Schöler, HR & Mitalipov, S 2014, 'Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos', Nature, vol. 508, no. 7498, pp. 101-104. https://doi.org/10.1038/nature13134
Kang E, Wu G, Ma H, Li Y, Tippner-Hedges R, Tachibana M et al. Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos. Nature. 2014;508(7498):101-104. https://doi.org/10.1038/nature13134
Kang, Eunju ; Wu, Guangming ; Ma, Hong ; Li, Ying ; Tippner-Hedges, Rebecca ; Tachibana, Masahito ; Sparman, Michelle ; Wolf, Don P. ; Schöler, Hans R. ; Mitalipov, Shoukhrat. / Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos. In: Nature. 2014 ; Vol. 508, No. 7498. pp. 101-104.
@article{e0476807d2ae40368610879e96473848,
title = "Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos",
abstract = "Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative applications, as donated or discarded embryos are more accessible than unfertilized MII oocytes.",
author = "Eunju Kang and Guangming Wu and Hong Ma and Ying Li and Rebecca Tippner-Hedges and Masahito Tachibana and Michelle Sparman and Wolf, {Don P.} and Sch{\"o}ler, {Hans R.} and Shoukhrat Mitalipov",
year = "2014",
doi = "10.1038/nature13134",
language = "English (US)",
volume = "508",
pages = "101--104",
journal = "Nature",
issn = "0028-0836",
publisher = "Nature Publishing Group",
number = "7498",

}

TY - JOUR

T1 - Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos

AU - Kang, Eunju

AU - Wu, Guangming

AU - Ma, Hong

AU - Li, Ying

AU - Tippner-Hedges, Rebecca

AU - Tachibana, Masahito

AU - Sparman, Michelle

AU - Wolf, Don P.

AU - Schöler, Hans R.

AU - Mitalipov, Shoukhrat

PY - 2014

Y1 - 2014

N2 - Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative applications, as donated or discarded embryos are more accessible than unfertilized MII oocytes.

AB - Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative applications, as donated or discarded embryos are more accessible than unfertilized MII oocytes.

UR - http://www.scopus.com/inward/record.url?scp=84899674216&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84899674216&partnerID=8YFLogxK

U2 - 10.1038/nature13134

DO - 10.1038/nature13134

M3 - Article

C2 - 24670652

AN - SCOPUS:84899674216

VL - 508

SP - 101

EP - 104

JO - Nature

JF - Nature

SN - 0028-0836

IS - 7498

ER -