Year 4

Our CIRM funded project have provided strong evidence suggesting microRNAs (miRNA), and in a broader perspective, non-coding RNAs, as essential gene regulators in the self-renewal and pluripotency of embryonic stem cells. Although non-coding RNAs, including miRNAs, do not have protein coding capacity, but they possess strong effects in regulating Using genomic approaches, embryonic stem cell culture and induced pluripotent stem cell culture and molecular biology, we were able to screen for, identify and characterize a number of non-coding RNAs, particularly, microRNAs, as important regulators for stem cell self-renewal and differentiation.Our results can have profound implications on the role of ncRNAs in the generation of induced pluripotent stem cells (iPSCs). Several major progresses have been made for the year 4 of our CIRM funded project. .

Last year, we have successfully carried out a functional screen to identify miRNAs with important roles in regulating ES cell self-renewal. Among the miRNA candidates emerged from this screen, we primarily focused on the functional and mechanistic characterization of several important miRNA regulators for ES cell pluripotency. We investigated roles of miR-34 miRNAs, whose deficiency significantly promotes somatic reprogramming. We studied the role of miR-34 in the epigenetic remodeling during somatic reprogramming. We also studied the function of mir-290-295 cluster, which constitute the majority of miRNA species in the ES cells. We generated ES cell deficient for the mir-290-295 miRNA cluster to characterize its effects on ES cell self-renewal, and we also explored the functional redundancy of the mir-290-295 family miRNAs by functionally characterized related miRNA family, including the mir17-92 miRNAs.

In the previous funding period, we also carried out a screen to identify miRNAs or non-coding RNA with important roles in regulating ES cell differentiation. Here, we functionally characterized the effects of miR-meso in mesoderm differentiation both in vitro and in vivo, using teratoma assays and KO ES cells. In addition, we identified a long ncRNA that exhibit an interesting pattern of expression during ES cell differentiation. We characterized its unique expression alteration during ES cell differentiation, and we are currently generating the knockout ES cells for this long ncRNA to further investigate its function. Finally, we characterized miRNA expression profiles during ES cell to Epiblast stem cell differentiation, and identified additional candidate miRNAs that regulate the exit of pluripotency of ES cells during their differentiation.

Finally, we successfully established the biological system to generate human iPSCs from human dermal fibroblasts, and will use this system to explore the role of specific ncRNAs in human somatic reprogramming. Our previous results using mouse ES cells and iPSCs have prepared us well in exploring the significance of our finding in human. This will be a main focus to achieve in our last funding period.