Plastic Surgery Research Council
Members Only  |  Contact  |  PSRC on Facebook
PSRC 60th Annual Meeting
Program and Abstracts

Back to 2015 Annual Meeting Program


TWIST1 Silencing Enhances in vitro and in vivo Osteogenic Differentiation of Human Adipose Derived Stem Cells by Triggering Activation of BMP-ERK/FGF Signaling and TAZ Upregulation.
Kshemendra Senarath-Yapa, MA MBBChir MRCS1, Natalina Quarto, PhD1, Andrea Renda, PhD2, Michael Longaker, MD MBA1.
1The Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University, Stanford, CA, USA, 2Dipartimento di Scienze Biomediche Avanzate, Universita’ degli Studi di Napoli. Federico II, Napoli, 80131 Italy, Napoli, Italy.

PURPOSE:Human adipose-derived stem cells (hASCs), found abundantly in lipoaspirate are an attractive source of cells for the field of regenerative medicine. Optimizing the osteogenic capacity of hASCs through modulation of signaling pathways that govern osteogenic capacity is a key objective with translational implications. This study investigates TWIST1 function in hASCs. TWIST1 is a transcription factor that plays roles in lineage commitment and differentiation and is also associated with Saethre-Chotzen syndrome in humans. Our results show that TWIST1 shRNA silencing increased the osteogenic potential of hASCs in-vitro and their skeletal regenerative ability when applied in-vivo. We demonstrate that the increased osteogenic capacity observed with TWIST1 knockdown in hASCs is mediated through endogenous activation of BMP and ERK/FGF signaling leading, in turn, to upregulation of TAZ, a transcriptional modulator of MSC differentiation along the osteoblast lineage.
METHODS:Human ASCs were obtained from lipoaspirates of patients following approved guidelines. Lipoaspirates were washed with PBS, digested with Collagenase at 37°C, and then centrifuged. The pellet was resuspended and plated. Knockdown of TWIST1 and also TAZ in hASCs was achieved with shRNA lentiviral particles. Osteogenic differentiation assays were performed using shTWIST1 hASCs, shTAZ hASCs and scramble (control) hASCs, respectively and qRT analysis performed for specific osteogenic markers. Cells (250,000) were suspended in 100μl FCS and seeded on (PLGA) scaffolds. Scaffolds were placed in 4mm calvarial defects in nude CD1 mice 2 hours after in vitro seeding. Treatment groups included no treatment (empty), scaffold with serum, scaffold seeded with shTWIST1 hASCs, scaffold seeded with scramble hASCs, scaffold seeded with shTWIST1/TAZ hASCs and scaffold seeded with shTAZ hASCs. Calvarial healing was assessed using μCT and histological analysis.
RESULTS:qRT PCR and immunoblotting of cell lysates revealed that endogenous expression of TWIST1 is downregulated in hASCs undergoing osteogenic differentiation. Twist expression was effectively knocked down using shRNA. Downregulation of endogenous TWIST1 increases the in-vitro osteogenic potential of hASCs as shown by increased mineralization on Alizarin red staining and significantly increased expression of osteogenic markers RUNX and OSX. Downregulation of endogenous TWIST1 enhances BMP and ERK1/2-FGF signaling pathways in hASCs. Upregulation of endogenous TAZ in shTWIST1 hASCs contributes to their enhanced osteogenic ability. shTWIST1 hASCs accelerate and potentiate bone repair in-vivo relative to control scrambled ASCs (P<0.05). Co-silencing of both TWIST1 and TAZ abrogated the effect elicited by TWIST1 knockdown alone.
CONCLUSION:We demonstrate that the increased osteogenic capacity observed with TWIST1 knockdown in hASCs is mediated through activation of BMP and ERK/FGF signaling leading, in turn, to upregulation of TAZ, a transcriptional modulator of mesenchymal stem cells differentiation along the osteoblast lineage. Inhibition either of BMP or ERK/FGF signaling suppressed TAZ upregulation and the enhanced osteogenesis in shTWIST1 hASCs. Co-silencing of both TWIST1 and TAZ abrogated the effect elicited by TWIST1 knockdown thus, identifying TAZ as a downstream mediator through which TWIST1 knockdown enhanced osteogenic differentiation in hASCs. Our functional study contributes to a better knowledge of molecular mechanisms governing the osteogenic ability of hASCs, and highlights TWIST1 as a potential target to facilitate in-vivo bone healing.


Back to 2015 Annual Meeting Program