Somatic Cell Reprogramming using microRNA



MicroRNAs (miRNA) are short 20- to 25-nucleotide (nt) long single-stranded noncoding RNAs that represent a class of small regulatory RNAs. Found in plants and animals, miRNAs regulate gene expression posttranscriptionally by triggering the degradation of target messenger RNAs (mRNA) and thus play an important role in a wide range of cellular processes. Biogenesis of a miRNA starts with pol-II/III-mediated synthesis of a primary miRNA transcript (pri-miRNA) characterized by a stem and loop structure. Pri-miRNAs are processesd in the nucleus by the ribonuclease Drosha to a shorter hairpin precursor miRNA (pre-miRNA) approximately 60-nt in length. After being transported into cytoplasm by Exportin-5, pre-miRNAs are further processesed into mature miRNAs by the cytoplasmic ribonuclease Dicer. MiRNAs associate with a number of proteins to form RNA-induced silencing complex (RISC) that bind with mRNAs with complementary sequences to the miRNAs. These target mRNAs are then cleaved and degraded and protein translations are inhibited. Recently a novel class of miRNAs was identified; they are unique in that their pri-miRNAs are derived from introns that are removed during RNA splicing following pol-II-mediated DNA transcription. These miRNAs are called intronic miRNAs.

Induced Pluripotent Stem Cells (iPS Cells)

Human embryonic stem (hES) cells are derived from human blastocysts and have the capacity to develop into any type of cells in our body. These types of cells are called pluripotent and they hold promising potentials in cell-based therapies and regenerative medicine. Concerns with the application of hES cells, particularly the issue of immune rejection stemming from incompatibility between the patient and donor cells, call for alternative approaches of generating pluripotent stem cells for therapeutic applications. Recently it was shown that retrovirus-mediated transfection of a combination of four transcription factors was able to ‘reprogram’ mouse fibroblast and human somatic cells to undifferentiated, pluripotent stem cells. These induced pluripotent stem or iPS cells exhibit the essential characteristics of ES cells as confirmed by morphological and other molecular criteria. Even though the iPS approach possesses a great therapeutic potential, the following concerns would have to be addressed for iPS technology to be practical for human therapy: the use of retrovirus for transfection, introduction of an oncogene (c-myc), and the technical difficulties involved in simultaneously transfecting four genes into cells.

MiRNA Reprogrammed Pluripoten Stem Cells (mirPS Cells)

microRNAs (miRNA) have been identified to play a critical role in ES cell maintenance and renewal. One of these miRNAs, miRNA-302 (mir-302), is found to be highly expressed in ES cells as compared to differentiated cells. So far mir-302s are known to target over 400 conserved genes in both human and mouse; these genes encode for proteins that function as developmental signals during differentiation processes in embryonic development. To test the hypothesis that mir-302s play an important role in driving and maintaining pluripotency in cells, a Pol-II-based intronic miRNA expression system was developed to transgenically express mir-302s in human cells. Pluripotent and ES-like cells have been successfully generated from human somatic and cancer cells by expressing mir-302s using this approach. These mir-302-regrogrammed pluripotent stem (mirPS) cells include human primary epidermal skin culture (hpESC), prostate carcinoma PC3 and LNCaP, breast carcinoma MCF-7 and Melanoma Colo cell lines. The pluripotent status of these mirPS cells has been confirmed by morphological criteria and their overexpression of a number of ES marker proteins. To further improve and realize the potentials of the mirPS technology, current efforts are focused on developing non-viral methods of transfecting mir-302s and to better understand how mirPS cells differentiate into various types of cells.

Mir-302 and mirPS® Cells

The Mir-302 family is expressed most abundantly in slow-growing human embryonic stem (hES) cells and quickly decreases after cells differentiate and proliferate. Transgenic delivery of mir-302s into human skin, hair follicle and melanoma cells has been shown to reprogram these somatic cell to reprogrammed pluripotent stem (iPS)-like cells1. The mir-302-reprogrammed pluripotent stem cells, namely mirPS cells, not only express many key hES cell markers, such as Oct4 and Sox2, but also have a highly demethylated genome similar to a reprogrammed zygotic genome. Microarray analyses have revealed that genome-wide gene expression patterns between the mirPS and hES H1 and H9 cells share over 86%–92% similarity. Under molecular guidance in vitro, these mirPS cells can differentiate into many distinct tissue cell types. Therefore, mir-302s may be used in place of Oct4, Sox2 and Nanog for more simple and efficient generation of iPS cell lines.