Journal Article

Osteogenic Programming of Human Mesenchymal Stem Cells with Highly Efficient Intracellular Delivery of RUNX2

Dixon, James E., Abu‐Awwad, Hosam Al‐Deen M., Thiagarajan, Lalitha

2017

Overview

Mesenchymal stem cells (MSCs) are being exploited in regenerative medicine due to their tri‐lineage differentiation and immunomodulation activity. Currently, there are two major challenges when directing the differentiation of MSCs for therapeutic applications. First, chemical and growth factor strategies to direct osteogenesis in vivo lack specificity for targeted delivery with desired effects. Second, MSC differentiation by gene therapy is difficult as transfection with existing approaches is clinically impractical (viral transfection) or have low efficacy (lipid‐mediated transfection). These challenges can be avoided by directly delivering nonvirally derived recombinant protein transcription factors with the glycosaminoglycan‐binding enhanced transduction (GET) delivery system (P21 and 8R peptides). We used the osteogenic master regulator, RUNX2 as a programming factor due to its stage‐specific role in osteochondral differentiation pathways. Herein, we engineered GET‐fusion proteins and compared sequential osteogenic changes in MSCs, induced by exposure to GET fusion proteins or conventional stimulation methods (dexamethasone and Bone morphogenetic protein 2). By assessing loss of stem cell‐surface markers, upregulation of osteogenic genes and matrix mineralization, we demonstrate that GET‐RUNX2 efficiently transduces MSCs and triggers osteogenesis by enhancing target gene expression directly. The high transduction efficiency of GET system holds great promise for stem cell therapies by allowing reproducible transcriptional control in stem cells, potentially bypassing problems observed with high‐concentration growth‐factor or pleiotropic steroid therapies. Stem Cells Translational Medicine 2017;6:2146–2159 Many regenerative medicine approaches employ the use of mesenchymal stem cells (MSCs) as they can be obtained directly from the patient from a number of tissues, can be expanded in culture, and have been shown to have positive clinical outcomes in a number of trials. These cells are multipotent meaning they have the ability to become different tissue‐type cells (fat, bone, cartilage) with a predisposition to convert into specific tissue types (differentiate) depending on the source tissue from which they were first isolated. Methods to control or change this predisposition will be key to exploiting them to repair tissue in cell therapies. Here, we describe a method to program the gene expression of MSCs to differentiate them efficiently into bone cells. Importantly this technique can overcome the predisposition to become alternatives (such as cartilage) directly at the level of gene expression. Our technology is based upon delivering a recombinant transcription factor protein (RUNX2) which does not genetically modify cells unlike gene therapy. This can now be exploited for programming MSCs when developing strategies for repairing bone trauma and disorders. 1. Schematic representation of directed osteogenesis of human MSCs using P21‐RUNX2‐8R. 2. Fluorescent images of human MSCs showing efficient delivery of P21‐RUNX2‐8R (Scale bar 50 μm). 3. Dual luciferase assay showing transcriptional activity of transduced P21‐RUNX28R. 4. Expression of osteogenic markers post transduction of P21‐RUNX2‐8R (Scale bar 20 μm). 5. Alizarin Red staining showing enhanced matrix mineralization using P21‐RUNX2‐8R (×1.5 magnification).

• Publisher: Oxford University Press,John Wiley and Sons Inc
• Subject: Bone Marrow Stem Cells / Bone morphogenetic protein 2 / Bone Morphogenetic Protein 2 - pharmacology / Cbfa-1 protein / Cell Culture Advances / Cell surface / Cells, Cultured / Cellular Reprogramming / Cellular Reprogramming Techniques - methods / Cell‐penetrating peptide / Core Binding Factor Alpha 1 Subunit - genetics / Core Binding Factor Alpha 1 Subunit - metabolism / Councils / Dexamethasone / DNA binding proteins / Ethylenediaminetetraacetic acid / Gene expression / Gene therapy / Gene Transfer Techniques / Genetic engineering / Glycosaminoglycan‐binding enhanced transduction / Growth factors / Health aspects / Humans / Immunomodulation / Intracellular transduction / Mechanisms of Differentiation / Medicine / Mesenchymal stem cells / Mesenchymal Stem Cells - cytology / Mesenchymal Stem Cells - drug effects / Mesenchymal Stem Cells - metabolism / Mesenchyme / Methods / Methylene blue / Mineralization / Osteogenesis / Peptides / Protein binding / Proteins / Recombinant proteins / Regenerative medicine / RUNX2 / Stem cells / Surface markers / Therapeutic applications / Tissue Engineering and Regenerative Medicine / Transcription factors / Transfection / Translational s and Reviews / Transplantation / Trauma