Non-Viral Engineering of Skin Precursor-Derived Schwann Cells for Enhanced NT-3 Production in Adherent and Microcarrier Culture

Abstract

Genetic engineering of stem cells and their derivatives has the potential to enhance their regenerative capabilities. Here, dendrimer- and lipofection-based approaches were used for non-viral neurotrophin-3 (NT-3) over-expression in Schwann cells differentiated from skin precursors (SKP-SCs). A variety of dendrimers were first tested for transfection efficiency on HEK 293T cells, with PAMAMNH2 G4 found most effective and used subsequently for SKP-SCs transfection. Plasmid-based expression resulted in increased NT-3 release from SKP-SCs in both adherent and microcarrier-based culture. In a proof-of-concept study, the microcarrier/SKP-SCs were implanted into the injured nerve, and transfected cells were shown to detach, integrate into the nerve tissue and associate with regenerating axons. Virus-free systems for transient neurotrophin expression are a feasible and biologically safe option to increase the therapeutic value of stem cells and stem cell-derived cells in nerve repair strategies. Further work to develop bioprocesses for expansion of SKP-SCs on microcarriers in bioreactors is still needed.

c-Jun Gene-Modified Schwann Cells: Upregulating Multiple Neurotrophic Factors and Promoting Neurite Outgrowth

“…[12][13][14] Recent studies have revealed that delivery of SCs to NCs is advantageous for nerve repair, particularly when using SCs that overexpress an NF, such as basic fibroblast growth factor (FGF-2), 15,16 glial cell line-derived neurotrophic factor (GDNF), 17,18 nerve growth factor (NGF), 19 brainderived neurotrophic factor (BDNF), 20 or neurotrophin-3 (NT-3). 21 However, an increasing number of studies have indicated that the application of multiple NFs, rather than a single type, holds greater therapeutic promise. 4,22,23 If we can modify a cell to upregulate multiple NFs, nerve regeneration and functional recovery might be greatly enhanced.…”

Schwann cells transduced with a lentiviral vector encoding Fgf‐2 promote motor neuron regeneration following sciatic nerve injury

“…Gene therapy is a relatively recent approach to study the regenerative effects of overexpression of specific therapeutic proteins on the injured nerve. The introduction of genetically engineered glia cells overexpressing trophic and tropic factors in nerve guides may improve their proregenerative properties and guides filled with genetically modified cells more closely resemble the autograft (Eggers et al, ; Godinho et al, ; Gravvanis et al, ; Hu et al, ; Kokai et al, ; Li et al, ; May et al, ; Santosa et al, ; Shakhbazau et al, , b; Yu et al, ). Adenoviruses were the first viral vectors used to successfully transduce Schwann cells in a peripheral nerve (Dijkhuizen et al, ; Shy et al, ).…”

Inter‐microcarrier transfer and phenotypic stability of stem cell‐derived Schwann cells in stirred suspension bioreactor culture

“…To facilitate cell adherence to microcarriers, cell/microcarrier suspensions were agitated intermittently for 3 min every half an hour for 5 h (Alfred et al, ) and continued in static or stirred culture. In this study, “Static Microcarrier culture” refers to SKP‐SCs‐seeded microcarriers cultured without stirring, whereas “Stirred Microcarrier culture” refers to cells cultured on microcarriers in a custom mini‐stirred culture model (vessel 50 mL size) with 10.0 mL SC medium described earlier (Shakhbazau et al, ) (5 × 10 4 cells/mL, 7400 cells/cm 2 on Cytodex 3 carriers) that was agitated at 60 rpm (Stirred Microcarrier). Cells were also cultured in dynamic culture in 100.0 mL SC medium in a stirred suspension bioreactor (Stirred Microcarrier, Bioreactor) (NDS Technologies, Inc., Vineland, NJ, nominal volume 125 mL).…”