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转译而非转染限制了临床相关的、基于外源性 mRNA 对人骨髓间充质干细胞α-4 整合素表达的诱导。

Translation, but not transfection limits clinically relevant, exogenous mRNA based induction of alpha-4 integrin expression on human mesenchymal stem cells.

机构信息

NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.

Fraunhofer-Institute for Cell Therapy and Immunology, Department of Cell Therapy, University of Leipzig, Leipzig, Germany.

出版信息

Sci Rep. 2017 Apr 24;7(1):1103. doi: 10.1038/s41598-017-01304-3.


DOI:10.1038/s41598-017-01304-3
PMID:28439079
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5430815/
Abstract

Mesenchymal stem cells (MSCs) represent promising resource of cells for regenerative medicine in neurological disorders. However, efficient and minimally invasive methods of MSCs delivery to the brain still have to be developed. Intra-arterial route is very promising, but MSCs are missing machinery for diapedesis through blood-brain barrier. Thus, here we have tested a mRNA-based method to induce transient expression of ITGA4, an adhesion molecule actively involved in cell extravasation. We observed that transfection with an ITGA4-mRNA construct bearing a conventional cap analogue (7-methylguanosine) failed to produce ITGA4 protein, but exogenous ITGA4-mRNA was detected in transfected MSCs. This indicates that not transfection, but rather translation being the major roadblock. Stabilization of ITGA4-mRNA with SSB proteins resulted in ITGA4 protein synthesis in HEK293 cells only, whereas in MSCs, satisfactory results were obtained only after using an anti-reverse-cap-analogue (ARCA). The presence of ITGA4 protein in MSCs was transient and lasted for up to 24 h after transfection. Membranous location was confirmed by flow cytometry of viable non-permeabilized cells using anti-ITGA4 antibody. The mRNA-based expression of itga4 transgene is potentially sufficient for diapedesis after intra-arterial delivery. To conclude, mRNA-based engineering of stem cells is a rapid and integration-free method and attractive from the perspective of potential future clinical application.

摘要

间充质干细胞(MSCs)是神经紊乱再生医学中有前途的细胞资源。然而,仍需开发将 MSCs 有效且微创地递送至大脑的方法。经动脉途径非常有前景,但 MSCs 缺乏通过血脑屏障的穿细胞能力。因此,我们在这里测试了一种基于 mRNA 的方法来诱导 ITGA4 的瞬时表达,ITGA4 是一种积极参与细胞外渗的黏附分子。我们观察到,用带有常规帽类似物(7-甲基鸟苷)的 ITGA4-mRNA 构建体进行转染未能产生 ITGA4 蛋白,但在转染的 MSCs 中检测到外源性 ITGA4-mRNA。这表明不是转染,而是翻译是主要的障碍。使用 SSB 蛋白稳定 ITGA4-mRNA 仅导致 HEK293 细胞中的 ITGA4 蛋白合成,而在 MSCs 中,仅在用抗反向帽类似物(ARCA)后才能获得满意的结果。在转染后最多 24 小时内,MSCs 中 ITGA4 蛋白的表达是短暂的。通过使用抗 ITGA4 抗体对活的未通透细胞进行流式细胞术,证实了膜定位。经动脉递送后,ITGA4 转基因的基于 mRNA 的表达可能足以穿细胞。总之,基于 mRNA 的干细胞工程是一种快速且无整合的方法,从潜在的未来临床应用的角度来看具有吸引力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/3a69ef4d93b0/41598_2017_1304_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/68b22aa0dd5b/41598_2017_1304_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/c42c1254305f/41598_2017_1304_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/18db71ca6c73/41598_2017_1304_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/ec5b214b32c1/41598_2017_1304_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/6d33163fba17/41598_2017_1304_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/97632618d425/41598_2017_1304_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/99d4498189ae/41598_2017_1304_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/3a69ef4d93b0/41598_2017_1304_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/68b22aa0dd5b/41598_2017_1304_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/c42c1254305f/41598_2017_1304_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/18db71ca6c73/41598_2017_1304_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/ec5b214b32c1/41598_2017_1304_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/6d33163fba17/41598_2017_1304_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/97632618d425/41598_2017_1304_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/99d4498189ae/41598_2017_1304_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f18a/5430815/3a69ef4d93b0/41598_2017_1304_Fig8_HTML.jpg

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[7]
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本文引用的文献

[1]
Human bone marrow mesenchymal stromal/stem cells: current clinical applications and potential for hematology.

Int J Hematol. 2016-2

[2]
Mesenchymal stem cells engineered to express selectin ligands and IL-10 exert enhanced therapeutic efficacy in murine experimental autoimmune encephalomyelitis.

Biomaterials. 2016-1

[3]
Therapeutic Effects of Mesenchymal Stem Cells for Patients with Chronic Liver Diseases: Systematic Review and Meta-analysis.

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Excess Integrins Cause Lung Entrapment of Mesenchymal Stem Cells.

Stem Cells. 2015-11

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Neurology. 2014-3-7

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