Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Identification of BMP-Responsive Long Noncoding RNAs in Pluripotent Cells
Kevin J. Paik, A.B.1, Kun Qu, Ph.D.2, Brian Hsueh, A.B.1, Eduardo A. Torre, B.S.2, Ryan A. Flynn, B.S.2, Michael T. Chung, B.S.1, Andrew Spencley, B.S.3, Kevin C. Wang, M.D., Ph.D.3, Joseph C. Wu, M.D., Ph.D.4, Michael T. Longaker, M.D., M.B.A.1, Howard Y. Chang, M.D., Ph.D.2, Derrick C. Wan, M.D.1.
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford, CA, USA, 2Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford, CA, USA, 3Department of Dermatology, Stanford, CA, USA, 4Department of Radiology and Department of Medicine, Division of Cardiology, Stanford, CA, USA.

PURPOSE:
Pluripotent stem cells hold tremendous promise for the study of human diseases and for the treatment of various tissue deficits in regenerative medicine. A key challenge in controlling cell fate toward lineage-specific differentiation, however, is minimizing the risk of teratoma formation. Previous research has highlighted the important role that epigenetic regulation plays in the specification and maintenance of cell fate. Furthermore, a growing body of literature has shown that a novel class of long noncoding RNAs (lncRNAs) are capable of effecting changes to the chromatin landscape through histone modification, thus being able to render genes transcriptionally active or silent. As Bone Morphogenetic Proteins (BMPs) have been found to be closely involved in the regulation of differentiation among pluripotent cells, the present study sought to identify specific lncRNAs responsive to BMP-2 in order to gain a clearer understanding of the epigenetic machinery responsible for controlling cell fate toward osteogenic differentiation.
METHODS:
Human induced pluripotent stem cells (iPSCs) were cultured on Matrigel-coated tissue culture dishes with mTeSR™1 medium. Cells were treated with BMP-2 (200ng/ml) and total RNA was harvested with Trizol at 0, 6, 12, 18, 24, and 48 hours. Samples were treated with Turbo DNase followed by Ribo-Zero magnetic beads for DNA and rRNA depletion respectively. Fragmentation with alkaline hydrolysis buffer and first strand synthesis and second strand synthesis were performed. 3’-A tailing and ligation to Illumina linkers allowed for gel size selection and sequencing with the Illumina HiSeq System. Paired-end reads were mapped to the human genome reference sequence hg19. Transcriptome assembly was performed with Cufflinks and transcriptomes were merged. Reads per kilobase of transcript per million mapped reads (RPKM) for known transcripts was calculated, and significantly differentially expressed transcripts were identified. Differentially expressed transcripts were then further narrowed down by Gene Ontology (GO) term analysis to identify functional transcripts related to modulation of pluripotency and skeletal development.
RESULTS:
To visualize genome-mapped data, results were uploaded to the UCSC Genome Browser. Importantly SOX2 expression was found to decrease with longer exposure to BMP-2, confirming decreased pluripotency. From 5566 significantly differentially expressed transcripts (protein-coding: 78%, lncRNAs: 4%, other: 18%), twelve annotated transcripts were determined following GO term analysis. Of these, the lncRNA at LOC100505806 showed unidirectional upregulation in response to BMP-2, which was confirmed by qRT-PCR. Subsequent studies will be done to manipulate this transcript and to assess for changes to pluripotent cells similar to BMP-2 exposure.
CONCLUSION:
The precise role of lncRNAs in regulating differentiation remains undefined, but with a better understanding the potential exists to redirect the developmental process in iPSCs, facilitating their use in regenerative medicine. By defining lncRNAs that effect BMP-mediated changes to chromatin states, it may be possible to manipulate regulatory networks of pluripotent cells to promote osteogenic differentiation.


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