Assessment of ability of human adipose derived stem cells for long term overexpression of IL-11 and IL-13 as therapeutic cytokines

Abstract

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells with the therapeutic effects that make them one of the best sources for cell therapy. In this study, we aimed to assess the ability of human ADSCs for constant expression of IL-11 and IL-13, simultaneously. In this study, the characterized hADSCs were transduced with a lentiviral vector (PCDH-513B) containing IL-11 and IL-13 genes, and the ability of long-term expression of the transgenes was evaluated by ELISA technique on days 15, 45 and 75 after transduction. Our results indicated a high rate of transduction (more than 90%) in the isolated hADSCs. Our data showed the highest rate of expression on days 75 after transduction which was 242.67 pg/ml for IL-11 and 303.6 pg/ml for IL-13 compared with 35.2 pg/ml and 35.6 pg/ml in untreated cells, respectively (p = 0.001). Besides, MTT assay showed transduction of hADSCs with lentiviral viruses containing IL-11 and IL-13 had no adverse effect on hADSCs proliferation (p-value = 0.89). Finally, we successfully constructed a hADSC population stably overexpressing IL-11 as the neurotrophic cytokine and IL-13 as the anti-inflammatory cytokine and this transduced cells can be used for further studies in EAE mice model.

Keywords: Human adipose derived stem cell (hADSC); Interleukin11; Interleukin13; Lentiviral vector; Overexpression.

Fig. 1

I Flow cytometric analysis. (A) Flow cytometry analysis for untreated hADSCs expressed high level of CD73 and CD105 as positive markers of and low level of CD45 and CD14 as negative markers of mesenchymal stem cells. (B) Flow cytometry analysis for transducted hADSCs showed that the transduced cell still was positive for CD73 and CD105 and negative for CD45 and CD14. II assessment of differentiation potential of human Adipose-derived stem cells (hADSCs) before and after transduction. A) The osteogenic differentiation potential of untreated and transduced hADSCs (A1 and A2, respectively) after 21 days culture in specific medium was confirmed with calcium deposition which stained with Alizarin red. A3) control group (100X magnification). B) The adipogenic The differentiation potential of untreated and transduced hADSCs (B1 and B2, respectively) after 21 days culture in specific medium was confirmed with lipid droplets which stained with Oil Red O (100X magnification)

Fig. 2

I The transfection efficiency of Lenti-X 293T cells with Co-transfection of the three vectors by Calcium phosphate precipitation method (×40). (A1) fluorescence microscopy of Lenti-X 293T cells 24 h after transfection showed a high level of green irradiation. (A2) Light microscopy of Lenti-X 293T cells 24 h after transfection. II Flow cytometry analysis, show that 13.1% of the Lenti-X 293T cells were successfully transduced by the virus. III Transduction of hADSCs. (A1) fluorescence microscopy of transducted hADSCs72h after transduction show a high level of GFP expression (×100). (A2) Light microscopy of transducted hADSCs72 h after transduction (×100). (B) Flow cytometry analysis of transduced hADSCs indicate that more than 90% of cells were expressing GFP

Fig. 3

I Evaluation of the effects of viral cytotoxicity on hADSC cells with MTT assay. Untreated hADSC and Mock cells use as the controls. MTT assay indicate no significant difference between transduced hADSCs and untreated hADSCs (p-value = 0.89). Also, the Mock cells has shown a high proliferation even more than untreated cells that indicated transduction with lentiviral vectors have no harmful effect on hADSCs (p-value = 0.59). II Confirmation of the Presence of the transgenes in transduced hADSCs as a 676 bp band

Fig. 4

I SDS page of transduced cells supernatant. Silver nitrate staining of a Poly acrylamide gel. A distinct band is found in the range of 15 and 20 kDa compared with untreated hADSCs which indicates the overexpression of IL-13 and IL-11 with molecular weights of 15.8 and 19.1 kDa, respectively. II ELISA of transduced cells supernatant. (A) Quantification of human IL-11 and IL-13 secreted by untreated hADSCs, MOCK cells and transduced hADSCs. Cell culture supernatants were collected after 15.45 and75 h of culture and IL-11 and IL-13 secretion determined by ELISA. Data represent mean ± SD of three independent samples assayed in triplicate. *p-value < 0.01,**p-value < 0.001, ***p-value < 0.0001. (A1) Overexpretion of IL-11 compared with the untreated and mock groups. Data show a significant increase of IL-11 on day 15 (0.0001) that is still significant on day 45 and 75 (0.001). (A2) Overexpretion of IL-13 secretion compared with the untreated and mock groups. Data indicate a significant high level of IL-13 secretion on day 15 and 45 (0.0001) and 75 (0.001)