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. Fall 2014;16(3):255-62.
Epub 2014 Oct 4.

Lentiviral Mediating Genetic Engineered Mesenchymal Stem Cells for Releasing IL-27 as a Gene Therapy Approach for Autoimmune Diseases

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Free PMC article

Lentiviral Mediating Genetic Engineered Mesenchymal Stem Cells for Releasing IL-27 as a Gene Therapy Approach for Autoimmune Diseases

Shohreh Hajizadeh-Sikaroodi et al. Cell J. .
Free PMC article

Abstract

Objective: Autoimmune diseases precede a complex dysregulation of the immune system. T helper17 (Th17) and interleukin (IL)-17 have central roles in initiation of inflammation and subsequent autoimmune diseases. IL-27 significantly controls autoimmune diseases by Th17 and IL-17 suppression. In the present study we have created genetic engineered mesenchymal stem cells (MSCs) that mediate with lentiviral vectors to release IL-27 as an adequate vehicle for ex vivo gene therapy in the reduction of inflammation and autoimmune diseases.

Materials and methods: In this experimental study, we isolated adipose-derived MSCs (AD-MSCs) from lipoaspirate and subsequently characterized them by differentiation. Two subunits of IL-27 (p28 and EBI3) were cloned in a pCDH-513B-1 lentiviral vector. Expressions of p28 and EBI3 (Epstein-Barr virus induced gene 3) were determined by real time polymerase chain reaction (PCR). MSCs were transduced by a pCDH-CMV-p28-IRES- EBI3-EF-copGFP-Pur lentiviral vector and the bioassay of IL-27 was evaluated by IL-10 expression.

Results: Cell differentiation confirmed true isolation of MSCs from lipoaspirate. Restriction enzyme digestion and sequencing verified successful cloning of both p28 and EBI3 in the pCDH-513B-1 lentiviral vector. Real time PCR showed high expressions level of IL-27 and IL-10 as well as accurate activity of IL-27.

Conclusion: The results showed transduction of functional IL-27 to AD-MSCs by means of a lentiviral vector. The lentiviral vector did not impact MSC characteristics.

Keywords: Autoimmune Disease; Gene Therapy; IL-27; Mesenchymal Stem Cells.

Figures

Fig 1
Fig 1
p28 and EBI3 genes inserted into the pCDH-513B-1 lentiviral vector. Digestion with XbaI-NotI showed an 8189 bp length of the pCDH-513B-1 backbone. The presence of a 1611 bp segment related to p28-IRES-EBI3and a segment of an 8189 bp related to pCDH-513B-1 confirmed that the cloning was established. A genetic map confirmed these data.
Fig 2
Fig 2
Transfection of HEK-293T for achieving viral particles. Panel A shows the HEK-293T culture. Panel B represents HEK- 293T at 18 hours after transfection by pCDH-CMV-p28-IRES-EBI3-EF1-copGFP-Pur. High expression of GFP in HEK-293T shows the high rate of transfection.
Fig 3
Fig 3
Isolation of mesenchymal stem cells (MSCs) from adipose tissue and characterized by differentiation. Panel A shows passage 2 adipose-derived MSCs (AD-MSCs), Panel B represents oil red staining of passage 2 AD-MSCs that differentiated into adipocytes. The vesicle that contained oil is visible in cells which showed adipogenic differentiation and Panel C shows the passage 2 AD-MSCs that were cultured in osteogenic differentiation medium and stained with alizarin red. Alizarin red stained the calcium deposits which confirmed osteogenic differentiation.
Fig 4
Fig 4
Transduction of adipocyte-derived mesenchymal stem cells (AD-MSCs) by lentiviral particles. Panel A shows AD-MSCs prior to transduction and Panel B shows transduced AD-MSCs by pCDH-CMV-p28-IRES-EBI3-EF1-copGFP-Pur lentiviral vector. The numerous green cells and GFP expression indicate a high level of transduction.
Fig 5
Fig 5
Expressions of IL-27, EBI3, and Oct-4 in adipose-derived mesenchymal stem cells (AD-MSCs) and transduced ADMSCs/ IL-27. Panel A shows the result of real time-PCR that confirmed the presence of a definite transcript by gel resolution. Expressions of p28 and EBI3 increased in transduced AD-MSCs/IL-27 compared with control AD-MSCs. Oct-4 expression did not show any significant difference. TBP was used as the RNA integrity control. Panel B represents the level of p28, EBI3 and Oct-4 expressions in AD-MSCs and AD-MSCs/IL-27 compared by real time PCR. The level of p28 expression increased 2000- fold, whereas EBI3 expression increased 650-fold.
Fig 6
Fig 6
T cell isolation and co-culture with COS-7. Panel A shows COS-7 cells prior to transduction. Panel B shows the transduced COS-7 by pCDH-CMV-p28-IRES-EBI3-EF1-copGFP-Pur lentiviral vector. COS-7 and COS-7/IL-27 were inactivated by mitomycin C. Panel C shows T cells isolated from the spleen of a C57BL/6 mouse.
Fig 7
Fig 7
Expression of IL10 as an IL-27 biological activity assay. Panel A shows the results of real time-PCR. IL-10 is overexpressed in T cell-COS7/IL-27 compared with T cell-COS7. GAPDH served as the endogenous reference gene. Panel B shows the level of IL10 expression in T cells cultured with COS-7/IL-27 increased 5-fold compared with COS-7 by real time PCR.

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