Three-dimensional microenvironmental priming of human mesenchymal stem cells in hydrogels facilitates efficient and rapid retroviral gene transduction via accelerated cell cycle synchronization

Bogyu Choi,Seunghee Oh,Yein Lee,Soo-Hong Lee,Hyungsuk Lee,James J. Moon,Yoshie Arai,Jinsung Ahn,Deogil Kim,Donyoung Kang

doi:10.1038/s41427-019-0127-9

Abstract

There are numerous approaches to improve the low transduction efficiency of retroviral vectors in two-dimensional (2D) cell culture substrates. However, the effect of a three-dimensional (3D) microenvironment, which better mimics in vivo conditions, is unknown. Cytocompatible hyaluronic acid (HA) hydrogels are a good candidate to study this issue. Here, photocrosslinkable HA hydrogels with an elastic modulus of 1.0–2.7 kPa were successfully prepared by varying the degree of methacrylation in the HA backbone. Culturing human adipose-derived stem cells (hASCs) in a 3D microenvironment significantly reduces the amount of time required for retroviral gene transduction compared with the conventional 2D method and maintains a high transduction efficiency. This acceleration of retroviral gene transduction correlates with the rate of cell-cycle synchronization. hASCs cultured in a 3D microenvironment have a shorter G1 phase and total cell-cycle length than hASCs cultured using the conventional 2D method. This cell-cycle regulation is dependent on expression of cyclin D1. In summary, prior culturing of hASCs in a 3D microenvironment accelerates retroviral gene transduction by regulating cyclin D1 expression and accelerating cell-cycle synchronization. We conclude that priming via culturing in a 3D microenvironment facilitates efficient and rapid retroviral gene transduction of hASCs without inducing apoptosis.

Highlights

Mesenchymal stem cells (MSCs), which can undergo self-renewal and are multipotent, have been extensively studied as therapeutic agents for various disorders due to their regenerative properties[1,2]
We employed photocrosslinkable hyaluronic acid (HA) hydrogels to investigate whether priming of primary human adiposederived stem cells via culturing in a 3D microenvironment affects the retroviral gene transduction efficiency
The Methacrylated HA (MAHA) solution (0.5% w/v in phosphate-buffered saline (PBS)) showed a rapid sol-to-gel transition after 10 s of UV irradiation in the presence of Irgacure 2959 (0.2% w/v), which was used as a photoinitiator (Fig. 1c)

Summary

Introduction

Mesenchymal stem cells (MSCs), which can undergo self-renewal and are multipotent, have been extensively studied as therapeutic agents for various disorders due to their regenerative properties[1,2]. According to a report by the National Institutes of Health in the United States, Widely used non-viral vector systems include cationic liposomes and polymers, nanoparticles, and electroporation. Calcium phosphate nanoparticles coated with highly cationic glutamine-conjugated oligochitosan significantly enhance the transfection of MSCs with Noggin-targeting small interfering RNA and osteogenesis of these cells[5]. Transfection of SOX5/SOX6/SOX9 and BMP-2 genes via electroporation enhances chondrogenesis and osteogenesis of MSCs, respectively[6,7]. Non-viral vectors are easy to use and do not elicit a marked immune response in the host, they have a low intracellular gene transfer efficiency and are not suitable for transfection of large populations of cells. Human stem cells transduced with retroviral vectors have been successfully used to treat patients with cancer and multiple congenital disorders[9,11]. The target cell specificity of retroviral vectors can be altered by pseudotyping with heterologous envelope proteins[13]

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Conclusion