Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate.
Fundamental behaviour such as cell fate, growth and death are mediated through the control of key genetic transcriptional regulators. These regulators are activated or repressed by the integration of multiple signalling molecules in spatio-temporal gradients. Engineering these gradients is complex but considered key in controlling tissue formation in regenerative medicine approaches. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor complexity but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly using GAG-binding domains to promote cell targeting, and cell penetrating peptides (CPPs) to allow cell entry. Herein we demonstrate that GET can be used in a three dimensional (3D) hydrogel matrix to produce gradients of intracellular transduction of mammalian cells. Using a compartmentalised diffusion model with a source-gel-sink (So-G-Si) assembly, we created gradients of reporter proteins (mRFP1-tagged) and a transcription factor (TF, myogenic master regulator MyoD) and showed that GET can be used to deliver molecules into cells spatio-temporally by monitoring intracellular transduction and gene expression programming as a function of location and time. The ability to spatio-temporally control the intracellular delivery of functional proteins will allow the establishment of gradients of cell programming in hydrogels and approaches to direct cellular behaviour for many regenerative medicine applications.



Regenerative medicine aims to reform functional biological tissues by controlling cell behaviour. Growth factors (GFs) are soluble cues presented to cells in spatio-temporal gradients and play important roles programming cell fate and gene expression. The efficient transduction of cells by GET (Glycosaminoglycan-enhanced transducing)-tagged transcription factors (TFs) can be used to by-pass GF-stimulation and directly program cells. For the first time we demonstrate diffusion of GET proteins generate stable protein transduction gradients. We demonstrated the feasibility of creating spatio-temporal gradients of GET-MyoD and show differential programing of myogenic differentiation. We believe that GET could provide a powerful tool to program cell behaviour using gradients of recombinant proteins that allow tissue generation directly by programming gene expression with TFs.
Wolfson Centre for Stem Cells, Tissue Engineering, and Modelling (STEM), Centre of Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK; Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt.
Hoda M
HM
Eltaher
Wolfson Centre for Stem Cells, Tissue Engineering, and Modelling (STEM), Centre of Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK.
Jing
J
Yang
Wolfson Centre for Stem Cells, Tissue Engineering, and Modelling (STEM), Centre of Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK. Electronic address: kevin.Shakesheff@nottingham.ac.uk.
Kevin M
KM
Shakesheff
Wolfson Centre for Stem Cells, Tissue Engineering, and Modelling (STEM), Centre of Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK. Electronic address: james.dixon@nottingham.ac.uk.
James E
JE
Dixon
D000818:Animals; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D019070:Cell Lineage; D004058:Diffusion; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005453:Fluorescence; D005786:Gene Expression Regulation / Q000187:drug effects; D006025:Glycosaminoglycans / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020100:Hydrogels / Q000494:pharmacology; D042541:Intracellular Space / Q000378:metabolism*; D051379:Mice; D024510:Muscle Development / Q000187:drug effects / Q000235:genetics; D017570:MyoD Protein / Q000378:metabolism; D041681:NIH 3T3 Cells; D013997:Time Factors; D014161:Transduction, Genetic
D006025:Glycosaminoglycans; D020100:Hydrogels; D017570:MyoD Protein; D016650:Fluorescein-5-isothiocyanate
CPP; Diffusion; GAG-binding enhanced transduction (GET); Gradient; Hydrogel; Intracellular transduction; Myogenesis