Fates of donor and recipient mitochondrial DNA during generation of interspecies SCNT-derived human ES-like cells.
To investigate nuclear donor and cytoplast recipient mitochondria fate and their effects on generation of interspecies somatic cell nuclear transfer (iSCNT)-derived human embryonic stem (ES)-like cells, iSCNT embryos were reconstructed between enucleated goat oocytes and human neural stem cells (hNSCs). A total of 10.74% cleaved embryos (13/121) developed to blastocyst stage. One typical primary ES-like (tpES-like) colony and two nontypical primary ES-like (non-tpES-like) colonies designated as non-tpES-like cell-1 and non-tpES-like cell-2, respectively, were obtained from the inner cell masses of iSCNT blastocysts. The tpES-like cells expressed ESC markers. Both human and goat mtDNA could be detected in the embryos at 2-8-, 16-32-cell, and blastocyst stages, and in tpES-like colony and two non-tpES-like colonies. Human mtDNA copies per cell from embryos at two- to eight-cell stage to the three colonies maintain almost its original level, whereas 2.88 x 10(5) goat mtDNA copies per oocyte decreased to 10.8 copies per tpES-like cell, 493 copies per non-tpES-like cell-1, and 77.6 copies per non-tpES-like cell-2, resulting in 43.75% (8.4/19.2), 1.24% (6.2/499), and 14.63% (13.3/90.9) mtDNA content in tpES-like cell, non-tpES-like cell-1, and non-tpES-like cell-2 was that of nuclear donor, respectively. Human-specific Tfam and Polg mRNA could be detected in cells of the three colonies. However, tpES-like colony failed to be passaged. The mRNA level of CoxIV encoded by nuclear donor in tpES-like cell was higher than that in non-tpES-like cell, but significantly lower than that of human ESC, suggesting proper nuclear-cytoplasmic communication would not be established in tpES-like cells. Thus, the data suggest that (1) goat oocytes could reprogram human neural stem cells (hNSCs) into embryonic state and further support the inner cell mass (ICM) of iSCNT blastocyst to form tpES-like colony; (2) nuclear donor mtDNA could be replicated and maintain its original level during the reduction of recipient mitochondrial DNA copies, (3) nuclear-cytoplasmic communication and recipient mtDNA copies might affect the derivation of iSCNT-derived ES-like cells.
Sha
Hong-ying
HY
Department of Neurosurgery, Fudan University Huashan Hospital, National Key Laboratory for Medical Neurobiology, Fudan University, Shanghai, People's Republic of China, 200040.
Chen
Jing-quan
JQ
Chen
Juan
J
Zhang
Peng-yue
PY
Wang
Pu
P
Chen
Lu-ping
LP
Cheng
Guo-xiang
GX
Zhu
Jian-hong
JH
D037881:Aborted Fetus; D000818:Animals; D053624:Blastocyst Inner Cell Mass / Q000166:cytology / Q000378:metabolism; D001921:Brain / Q000166:cytology; D002454:Cell Differentiation; D065150:Cellular Reprogramming / Q000235:genetics; D000074002:DNA Polymerase gamma; D004272:DNA, Mitochondrial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D022761:Gene Transfer, Horizontal / Q000235:genetics*; D006041:Goats; D006801:Humans; D008297:Male; D024101:Mitochondrial Proteins / Q000235:genetics; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics; D014180:Transplantation
D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C499631:TFAM protein, human; D014157:Transcription Factors; D003576:Electron Transport Complex IV; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human