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
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