This invention generally relates to a method for developing, generating and selecting human embryonic stem (hES)-like pluripotent cells using transgenic expression of intronic
microRNA-like
RNA agents. More particularly, the present invention relates to a method and composition for generating a non-naturally occurring
intron and its intronic components capable of being processed into mir-302-like
RNA molecules in mammalian cells and thus inducing certain specific
gene silencing effects on differentiation-related and fate-
determinant genes of the cells, resulting in
reprogramming the cells into a pluripotent embryonic stem (ES)-
cell-like state. The ES-like cells so obtained are strongly express hES
cell markers, such as Oct3 / 4, SSEA-3 and SSEA-4, and can be guided into various
tissue cell types by treating certain hormones and / or growth factors under a feeder-free
cell culture condition
in vitro, which may be used for
transplantation and
gene therapies. Therefore, the present invention offers a simple, effective and safe
gene manipulation approach for not only
reprogramming somatic cells into ES-like pluripotent cells but also facilitating the maintenance of pluripotent and renewal properties of ES cells under a feeder-free
cell culture condition, preventing the tedious retroviral
insertion of four large
transcription factor genes into one single cell as used in the previous iPS methods.