Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Oct 3;122(14):e23-32.
doi: 10.1182/blood-2013-04-495119. Epub 2013 Aug 26.

mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation

Oren Levy  1 , Weian Zhao, Luke J Mortensen, Sarah Leblanc, Kyle Tsang, Moyu Fu, Joseph A Phillips, Vinay Sagar, Priya Anandakumaran, Jessica Ngai, Cheryl H Cui, Peter Eimon, Matthew Angel, Charles P Lin, Mehmet Fatih Yanik, Jeffrey M Karp
Affiliations

mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation

Oren Levy et al. Blood. .

Abstract

Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy to treat several diseases and are compelling to consider as vehicles for delivery of biological agents. However, MSCs appear to act through a seemingly limited "hit-and-run" mode to quickly exert their therapeutic impact, mediated by several mechanisms, including a potent immunomodulatory secretome. Furthermore, MSC immunomodulatory properties are highly variable and the secretome composition following infusion is uncertain. To determine whether a transiently controlled antiinflammatory MSC secretome could be achieved at target sites of inflammation, we harnessed mRNA transfection to generate MSCs that simultaneously express functional rolling machinery (P-selectin glycoprotein ligand-1 [PSGL-1] and Sialyl-Lewis(x) [SLeX]) to rapidly target inflamed tissues and that express the potent immunosuppressive cytokine interleukin-10 (IL-10), which is not inherently produced by MSCs. Indeed, triple-transfected PSGL-1/SLeX/IL-10 MSCs transiently increased levels of IL-10 in the inflamed ear and showed a superior antiinflammatory effect in vivo, significantly reducing local inflammation following systemic administration. This was dependent on rapid localization of MSCs to the inflamed site. Overall, this study demonstrates that despite the rapid clearance of MSCs in vivo, engineered MSCs can be harnessed via a "hit-and-run" action for the targeted delivery of potent immunomodulatory factors to treat distant sites of inflammation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Improving MSC therapeutic potential via mRNA transfection with homing ligands and immunomodulatory factors. Illustration of (A) mRNA-engineered MSCs that express a combination of homing ligands (PSGL-1 and SLeX) and an immunomodulatory factor (IL-10), and (B) targeting mRNA-engineered MSCs to site of inflammation.
Figure 2
Figure 2
MSC expression of PSGL-1, SLeX, and IL-10 following mRNA transfection. (A) MSCs were transfected with either PSGL-1 (top) or FUT7 (bottom) and 24 hours later, flow cytometry analysis detected surface expression of PSGL-1 and SLeX, respectively. (B) After MSC transfection with PSGL-1 or FUT7, surface expression of PSGL-1 and SLeX, respectively, was confirmed for up to 7 days. Mean intensity of transfected cell population was normalized to respective isotype controls. Mean ± SD (n = 3). (C) Coexpression of PSGL-1 and SLeX from simultaneous transfection with PSGL-1 and FUT7 mRNA. (D) IL-10 secretion from native and IL-10 mRNA-transfected MSCs. Mean ± SD (n = 3). (E) IL-10 mRNA-transfected MSCs exhibit improved immunosuppressive effects in vitro. Resting human CD4+ T cells were cocultured with native or IL-10-transfected MSCs in the presence of CD3/CD28 Dynabeads, and CD4+ T cell proliferation was measured (7-AAD+/BrdU+ cells were defined as proliferating cells). *P < .05, 1-way ANOVA using Tukey’s HSD; error bars represent SD (n = 3).
Figure 3
Figure 3
PSGL-1/SLEX MSCs exhibit a robust rolling response on P-selectin-coated substrates in vitro and on inflamed endothelium in vivo. (A) Representative images showing PSGL-1/SLeX MSCs (yellow arrows) roll on a P-selectin surface in vitro at a substantially lower velocity than a native MSC. Shear stress in these images: 0.75 dyn/cm2. (B) Simultaneous expression of both PSGL-1 and SLeX is required to induce robust rolling of MSCs on P-selectin surface. Data are shown as mean velocity (calculated from 20 cells per group) ± SD. (C) Representative in vivo confocal microscopy images show a rolling (yellow arrows) and adhered (white arrows) PSGL-1/SLeX MSCs. (D) Histogram showing a representative velocity distribution of native MSCs and PSGL-1/SLeX MSCs on inflamed ear endothelium in vivo (representative analyzed population; velocity was calculated for at least 50 cells per group). Vcrit calculated as described in the “Methods” section.
Figure 4
Figure 4
PSGL-1/SLeX MSCs exhibit enhanced homing to healthy and γ-irradiated mouse BM. (A) Representative images of native MSCs (blue, DiD) and PSGL-1/SLeX MSCs (green, DiI) observed in both healthy and γ-irradiated BM (60× magnification; red, blood, rhodamine-dextran). (B) Quantitative analysis of MSC homing to nonirradiated or γ-irradiated BM. PSGL-1/SLeX MSCs exhibit enhanced homing to both healthy and γ-irradiated BM vs native MSCs (*P < .05, 1-way ANOVA using Tukey’s HSD; error bars represent ± standard error of the mean [SEM] [n = 4 per group]).
Figure 5
Figure 5
Incorporation of the PSGL-1/SLeX rolling machinery promotes rapid homing of MSCs to inflamed ear pinna. (A) Representative images of native MSCs (blue, DiD) and PSGL-1/SLeX MSCs (green, DiI) in healthy and inflamed mice ears (red, blood, rhodamine-dextran). (B) Quantitative analysis of MSC homing to the inflamed ear. Transfected MSCs exhibit statistically significant enhanced homing to the LPS-induced inflamed ear vs native MSCs at 2 hours postinjection (*P < .05, 1-way ANOVA using Tukey’s HSD; error bars represent ± SEM) (n = 4 and n = 7 for 2 hours and 24 hours, respectively). (C) A direct comparison between PSGL/SLeX MSCs and PSGL-1/SLeX/IL-10 MSCs reveals similar homing to inflamed ear pinna (ns = no statistical difference observed between the 2 groups at each time point; 1-way ANOVA using Tukey’s HSD; error bars represent ± SD, n = 4). Rapid clearance of MSC is observed, with peak number of cells observed at 24 to 48 hours and a significant decrease observed at 72 hours after cell administration (stack dimensions are 474 × 488 microns; *P < .05, 1-way ANOVA using Tukey’s HSD).
Figure 6
Figure 6
PSGL-1/SLeX/IL-10 MSCs display improved antiinflammatory impact in vivo via targeted delivery of IL-10 to the inflamed site. (A) Triple transfected PSGL-1/SLeX/IL-10 MSCs exhibit an antiinflammatory effect in vivo demonstrated via ear thickness measurements. Data are presented as decrease in inflamed ear thickness (after treatment with the different MSC groups) in comparison with control group (mice receiving saline treatment). *P < .05, 1-way ANOVA using Tukey’s HSD; error bars represent SEM) (n = 5 per group). (B) Only triple-transfected MSCs deliver a significant amount of IL-10 to the inflamed ear. At the specified time points following MSC transplantation, mice were sacrificed; ears were harvested and analyzed for presence of human IL-10 using an ELISA assay. Each point in the graph represents data from a single mouse. *P < .05 vs all other groups at the same time point, 1-way ANOVA using Tukey’s HSD; error bars represent SD (n = 4 per group).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315–317. - PubMed
    1. Singer NG, Caplan AI. Mesenchymal stem cells: mechanisms of inflammation. Annu Rev Pathol. 2011;6:457–478. - PubMed
    1. Hoogduijn MJ, Popp F, Verbeek R, et al. The immunomodulatory properties of mesenchymal stem cells and their use for immunotherapy. Int Immunopharmacol. 2010;10(12):1496–1500. - PubMed
    1. Liang J, Zhang H, Hua B. Allogeneic mesenchymal stem cells transplantation in treatment of multiple sclerosis. Mult Scler. 2009;15(5):644–646. - PubMed
    1. Alex P, Zachos NC, Nguyen T, et al. Distinct cytokine patterns identified from multiplex profiles of murine DSS and TNBS-induced colitis. Inflamm Bowel Dis. 2009;15(3):341–352. - PMC - PubMed

Publication types