mRNA Transfection of CXCR4-GFP Fusion—Simply Generated by PCR—Results in Efficient Migration of Primary Human Mesenchymal Stem Cells

Abstract

We present a general, entirely PCR-based strategy to construct mRNAs coding for green fluorescent protein (GFP) fusion proteins from a cDNA pool. We exemplify our approach for the chemokine receptor CXCR4. mRNA transfection of the PCR-generated fusion of CXCR4-GFP into K562 cells or primary mesenchymal stem cells (MSCs) resulted in excellent viability (> 90%) with more than 90% of target cells expressing easily detectable CXCR4-GFP for > 72 h. The fusion protein was localized in the plasma membrane and was rapidly internalized upon incubation with the CXCR4 ligand stromal cell-derived factor-1 (SDF-1). Transwell migration experiments showed significantly increased migration of CXCR4-GFP mRNA-transfected MSCs toward a gradient of SDF-1, demonstrating that mRNA-mediated chemokine receptor overexpression allows for transient initiation of chemotaxis. The presented strategy to construct a PCR-based fluorescent fusion protein can be generally applied to other genes of interest to study their function by simple overexpression and easy detection in primary cells.

Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

“…To optimize for stability, the mRNA cargo synthesized by in vitro translation was modified through 5= capping with ARCA and by 3= mRNA capping using poly(A) chains in cis and in trans (31). After transfection with ARCA-A100 Luc-mRNA, CD34-derived mouse dendritic cells (JAWSII cells) showed detectable luciferase activity within 1 h, which maximized at 8 h and declined 20-fold at 30 h (31), whereas transfection of primary human mesenchymal stem cells with a CXCR4-GFP fusion mRNA resulted in CXCR4 expression for up to 72 h and in efficient cell migration (32). To increase mRNA stability (33), our constructs included two sequential human beta-globin untranslated regions (UTRs).…”

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

“…Ryser and colleagues reported that electroporation of CXCR4-mRNA resulted in over 90% positively transfected MSCs 36 . However, electroporation disintegrates cell membrane stability with potential side effects and can reduce cell viability.…”

“…Thus, we decided to follow this strategy and engineer MSCs to overexpress the itga4 gene. Since viral-based methods have unfavourable safety profiles and DNA transfection is inefficient, we decided to utilize mRNA which was shown as an effective tool for genetic cell engineering 35, 36 . Since mRNA transfection is an integration-free method, inducing short-lasting expression and high transfection efficiency, it is ideal for the targeted transient induction of itga4 gene expression in MSCs.…”

Molecular Mechanisms Involved in Mesenchymal Stem Cell Migration to the Site of Acute Myocardial Infarction

“…Therefore, it may be the lack of surface expression of CXCR4 by MSC that leads to low efficiency of migration towards CXCL12. This has been further supported by the observation that enforced surface expression of CXCR4 leads to increased MSC engraftment and functional recovery after AMI [49, 69, 70]. Shi et al [65] investigated induced upregulation of CXCR4 in migration of Flk1 + MSC derived from human fetal bone marrow.…”