Electroporation-mediated Transfer of Runx2 and Osterix Genes to Enhance Osteogenesis of Adipose Stem Cells

Lee, JS; +Im, GI

+ Department of Orthopaedics, University of Dongguk Ilsan Hospital, Goyang, Korea

gunil@duih.org

INTRODUCTION: Adult stem cells are the promising potential for differentiation into

several cell types and predominantly the adipose stem cells (ASCs)

obtained from lipoaspirates has the multi-lineage prospective to

differentiate into various cell types. Several explorations have shown

that ASCs have the potential to differentiate into osteogenic lineages by

the transfection of BMP expression vectors. The constraint for the use of

BMP expression has low efficiency of its expression in the exogenous in

vivo system during osteogenesis. To address these facts several

researchers have explored the use of alternative bone specific

transcription factors to induce efficient osteogenesis. Transfection of

Runx2 and osterix in mesenchymal stem cells leads to the development

of osteoblastic cells and bone formation. However the foremost negative

aspect of viral transfection methods are immunogenicity and

mutagenesis for these reasons much effort has been made to go for

advantageous nonviral transfection by electroporation method to transfer

the growth factor genes. In the present study, we tested the hypothesis

that electroporation-mediated transfer of Runx2 and Osterix genes to

provoke in vitro and in vivo osteogenic potential in ASCs

METHODS: To create non-viral expressing RUNX-2, Osterix, full-length human RUNX-2, Osterix complementary DNA (cDNA) was amplified by

polymerase chainreaction (PCR) and cloned into pEGFPC1 mammalian

expression vector (Clontech, Palo Alto, CA). The microporatorTM

(Invitrogene) and the buffer system were utilized for gene delivery.

After microporation, the osteogenic differentiation was carried out using

2.5 x 105 ATMSCs in osteogenic medium. After days 7, cells were

analyzed for real time PCR, western blotting, ALP assay and alizarin-red

staining. To investigate in vivo ostegenesis of transfected ASCs, ASCs

were seeded in a PLGA scaffold, and the ASC-scaffold hybrid was

implanted in the subcutaneous tissue of nude mice. Six weeks after

implantation, the ASC-scaffold hybrid was harvested and analyzed by 3-

dimensional micro-CT, and also by histology.

RESULTS: To confirm the protein expression of RUNX-2, Osterix, we attempted to

western blotting (Fig 1). Expression of the Osterix gene was

significantly increased by RUNX-2, Osterix overexpression. Real-time

PCR analysis showed that the expression of OCN, ALP, Col1A1, and

BSP increased several fold in ATMSCs to which RUNX-2, Osterix

genes were transferred (Fig 2). RUNX-2, Osterix overexpression

induced ALP activity in ATMSCs. Alizarin-red staining demonstrated

that ATMSCs to which the RUNX-2, Osterix genes were transferred

exhibited greater accumulation of the calcium contents than negative

control (Fig 3). ASCs transfected with Runx2, Osterix, or both genes

demonstrated extensive mineralization. Histologic examination also

corroborated the findings from micro-CT (Fig 4).

Figure 1. Runx2 and Osterix gene and protein expression in tranfected

ASCs. (A): Transfected ASCs cultured in with OM medium were

harvested for real-time PCR assays for Runx2 and Osterix at days 7 and

14. (B): Western blot analysis for Runx2 and Osterix was performed at

day 7. GAPDH was used as the reference protein. *, p <0.05, n =3.

Figure 2. Gene expression of osteogenic differentiation markers in the

transfected ASCs. ALP,OCN, COL1A1, and BSP gene expression was

investigated by real-time PCR analysis 7 and 14 days after transfection

with Runx2, Osterix, or both. *, p <0.05, n =3

Figure 3. Protein expression of osteogenic differentiation markers and

mineralization in the transfected ASCs. (A): Detection of BSP, type I

collagen, and OCN 7 and 14 days after transfection with Runx2, Osterix,

or both using Western blotting. (B): Alkaline phosphates staining (C):

Alizarin Red (D): von Kossa staining

Figure 4. Bone formation and mineralization after in vivo implantation

of ASC-PLGA hybrids into the dorsal subcutaneous spaces of nude mice

for 6 weeks. Micro-computed tomographs of ASC-scaffold complex

implanted into nude mice for 6 weeks. Newly formed bone appears as

white dots white on the CT images and Masson’s trichrome staining.

DISCUSSION: The nonviral method for gene transfer shown here demonstrated a high

efficiency not preceded by other studies. Gene transfer of RUNX-2,

Osterix was effective in promoting osteogenesis. This nonviral gene

transfer system for RUNX-2, Osterix provides potent new means to

achieve osteogenic differentiation from ATMSCs.

ACKNOWLEDGEMENTS: This work was supported by a grant from the Korea Ministry of E

ducation, Science and Technology (Grant No 2010-0000305).

Poster No. 1740 • ORS 2011 Annual Meeting