In vitro osteogenic differentiation of adipose stem cells after lentiviral transduction with green fluorescent protein

Abstract

Background: Adipose-derived stem cells (ASCs) have the potential to differentiate into osteogenic cells that can be seeded into scaffolds for tissue engineering for use in craniofacial bone defects. Green fluorescent protein (GFP) has been widely used as a lineage marker for mammalian cells. The use of fluorescent proteins enables cells to be tracked during manipulation such as osteogenic differentiation within three-dimensional scaffolds. The purpose of this study was to examine whether ASCs introduced with GFP-encoding lentivirus vector exhibit adequate GFP fluorescence and whether the expression of GFP interfered with osteogenic differentiation of ASCs in both monolayer and three-dimensional scaffolds in vitro.

Methods: Primary ASCs were harvested from the inguinal fat pad of Sprague Dawley rats. Isolated ASCs were cultured and infected with a lentiviral vector encoding GFP and plated into both monolayers and three-dimensional scaffolds in vitro. The cells were then placed in osteogenic medium. Osteogenic differentiation of the GFP-ASCs was assessed using alizarin red S, alkaline phosphate staining, and immunohistochemistry staining of osteocalcin with quantification of alizarin red S and osteocalcin staining.

Results: The efficacy of infection of ASCs with a lentiviral vector encoding GFP was high. Cell-cultured GFP-ASCs remained fluorescent over the 8 weeks of the study period. The GFP-ASCs were successfully induced into osteogenic cells both in monolayers and three-dimensional scaffolds. Whereas the quanitification of alizarin red S revealed no difference between osteoinduced ASCs with or without GFP, the quantification of osteocalcin revealed increased staining in the GFP group.

Conclusions: Transduction of isolated ASCs using a lentiviral vector encoding GFP is an effective method for tracing osteoinduced ASCs in vitro. Quantification data showed no decrease in staining of the osteoinduced ASCs.

FIGURE 1

Green fluorescent protein fluorescence in GFP-ASCs 48 hours and 8 weeks after lentivirus infection. Light photomicrograph (A) and fluorescence photomicrograph (B) of GFP-ASCs cultured for 48 hours after infection with lentiviral vector. More than 90% of the ASCs were positive for GFP expression. Light photomicrograph (C) and fluorescence photomicrograph (D) of ASCs cultured for 8 weeks after infection with lentiviral vector. The GFP fluorescence is still easily detected. (Original magnification × 100.)

FIGURE 2

A, GFP-ASCs were incubated in the control medium for 3 weeks and showed an elongated fibroblastic appearance. B, Fluorescence microscopic images. C, After culture under osteogenic differentiation conditions for 3 weeks, the GFP-ASCs aggregated together in islands. D, Fluorescence microscopic images. (Original magnification × 100.)

FIGURE 3

Confirmation of the presence of ASCs by GFP fluorescence (GFP) and osteogenic differentiation of GFP-ASCs, AR staining, AP, and immunohistochemistry for osteocalcin (OC). Cells were cultured either in the control medium (CM) or the osteogenic medium (OM) for 4 weeks. (Original magnification × 200.)

FIGURE 4

Distribution of ASC-GFP in the three-dimensional Gelfoam scaffold 48 hours and 2 weeks after the viral infection and under fluorescence microscopy. A and B, Forty-eight hours after the inoculation of the cells. C and D, Two weeks after osteoinduction, increased GFP-positive cells were observed. (Original magnification × 100.)

FIGURE 5

Immunohistochemical staining for osteocalcin in the GFP-ASCs inside the Gelfoam scaffold. A, The GFP-ASC scaffold construct cultured in normal medium. B, The GFP-ASC scaffold construct cultured in osteogenic medium. (Original magnification of all images × 200.)

FIGURE 6

Comparison of AR S–stained calcium nodules in GFP-negative and GFP-positive ASCs in cell culture 4 weeks after osteoinduction in two-dimensional culture.

FIGURE 7

Comparison of the osteocalcin expression of the GFP-negative and the GFP-positive ASCs in cell culture 4 weeks after osteoinduction in two-dimensional culture. There is a significant difference between the 2 groups (*P < 0.05).