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.