Abstract

Over-expression of miR-361 in Embryonic Stem Cells as a Means to Generate Clinically-Relevant Osteo-Progenitors

Devon Duron Ehnes, Graduate Student, University of California-Riverside

Low bone density is a rapidly growing problem; the U.S. Surgeon General predicts that by 2020, over half of all Americans will have weak bones. The advent of tissue engineering from embryonic stem cells has begun to provide viable solutions and therapies for formerly devastating diseases and injuries in a variety of organs. However, due to the molecular intricacy and limited understanding of bone development, scientists have yet to pinpoint a feasible way to apply these newfound therapies to patients with low bone mass.
The discovery of the microRNA (miRNA, miR) and its implication as a key regulator of protein expression during embryonic development has opened up many possibilities as to the manipulation of stem cell differentiation. In order to identify miRNAs that regulate the specification of osteogenic precursors, our lab previously conducted a miRNA screen using osteogenically differentiating ESCs. From 25 differentially regulated miRNAs, miR-361 stood out, as its overexpression resulted in the largest increase in ESC calcification, a hallmark of maturing osteogenic cultures.  In order to elucidate the role of miR-361 in modulating osteogenesis, we identified prickle1/2 as a direct target of miR-361. As an upstream negative regulator of Dishevelled (Dvl), prickle is implicated in convergent extension movements during neurulation by affecting beta-catenin (CatnB). Western blots and immunocytochemistry suggested that overexpression of miR-361 in ESCs achieved a downregulation of nuclear CatnB levels concomitant with an up-regulation of Dvl1 levels. This occurred at a time point of differentiation at which mesenchyme is specified from neural crest cells that have the capability to subsequently differentiate into osteoprogenitors. In conclusion, overexpression of miR-361 may be exploited in vitro to enhance the efficiency of directed differentiation of pluripotent stem cells into osteoprogenitors, which then may be employed clinically to treat patients wi