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Method Article
Dieter Egli
The New York Stem Cell Foundation Research Institute
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium from the medium can improve developmental potential. 2) and on the insight that an efficient stimulus for oocyte activation is required; 3) and that histone deacetylase inhibitors facilitate reprogramming after nuclear transfer. A realistic expectation is that this protocol will result in about 10% of the oocytes developing to the blastocyst stage, most of which will be suitable for stem cell derivation. While most aspects of the protocol can be well controlled, variation in developmental efficiency when using oocytes of different donors should be expected.
Note: this protocol is a guideline that allows ES cell derivation after somatic cell nuclear transfer. The author strongly discourages the use of this protocol or aspects of it in attempts for human reproductive cloning.
Keywords
stem cells, nuclear transfer, calcium, fusion, blastocyst, embryonic stem cell (ES) cell line
Figure 1
Figure 2
the authors declare no competing financial interests
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.pdf
STR profile example example of an STR profile the STR profile is of our adult somatic cell nuclear transfer ES cell line from a type 1 diabetic. A match to 1018 hFb p4 (CLG-6537), the somatic donor cell line is stated. Such a match, together with a diploid karyotype is unambigious proof that the cell line derived is of somatic origin.
- supplement0.pdf
Protocol as pdf file Protocol for printing
- supplement0.mov
Movie Preparation of capillaries.
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Version History
CURRENT STATUS: Posted
Version 1
Posted 28 Apr, 2014
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Subject Areas
Cell biology
Associated Publications
Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells
by Mitsutoshi Yamada, Bjarki Johannesson, Ido Sagi, +12
Nature (07 April, 2014)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Dieter Egli
The New York Stem Cell Foundation Research Institute
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 28 Apr, 2014
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Cell biology
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium from the medium can improve developmental potential. 2) and on the insight that an efficient stimulus for oocyte activation is required; 3) and that histone deacetylase inhibitors facilitate reprogramming after nuclear transfer. A realistic expectation is that this protocol will result in about 10% of the oocytes developing to the blastocyst stage, most of which will be suitable for stem cell derivation. While most aspects of the protocol can be well controlled, variation in developmental efficiency when using oocytes of different donors should be expected.
Note: this protocol is a guideline that allows ES cell derivation after somatic cell nuclear transfer. The author strongly discourages the use of this protocol or aspects of it in attempts for human reproductive cloning.
Figure 1
Figure 2
the authors declare no competing financial interests
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.pdf
STR profile example example of an STR profile the STR profile is of our adult somatic cell nuclear transfer ES cell line from a type 1 diabetic. A match to 1018 hFb p4 (CLG-6537), the somatic donor cell line is stated. Such a match, together with a diploid karyotype is unambigious proof that the cell line derived is of somatic origin.
- supplement0.pdf
Protocol as pdf file Protocol for printing
- supplement0.mov
Movie Preparation of capillaries.
Loading...
Associated Publications
Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells
by Mitsutoshi Yamada, Bjarki Johannesson, Ido Sagi, +12
Nature (07 April, 2014)