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Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate.

Hoda M Eltaher1, Jing Yang2, Kevin M Shakesheff2

  • 1Wolfson 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.

Acta Biomaterialia
|June 7, 2016
PubMed
Summary

We developed GAG-binding enhanced transduction (GET) to create protein gradients within 3D hydrogels, enabling precise control over cell programming for regenerative medicine applications.

Keywords:
CPPDiffusionGAG-binding enhanced transduction (GET)GradientHydrogelIntracellular transductionMyogenesis

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Area of Science:

  • Biotechnology
  • Regenerative Medicine
  • Cell Biology

Background:

  • Cell fate, growth, and death are controlled by genetic transcriptional regulators activated by signaling molecules in spatio-temporal gradients.
  • Engineering these gradients is crucial for controlling tissue formation in regenerative medicine.
  • Direct cell programming using transcription factors bypasses growth factor complexity but requires spatio-temporal delivery.

Purpose of the Study:

  • To demonstrate the use of GAG-binding enhanced transduction (GET) technology to create intracellular protein gradients within a 3D hydrogel matrix.
  • To show that GET can achieve spatio-temporal control of protein delivery and subsequent gene expression programming.
  • To establish a novel method for directing cellular behavior in regenerative medicine.

Main Methods:

  • Utilized a compartmentalized diffusion model (source-gel-sink) within a 3D hydrogel.
  • Employed GET technology with GAG-binding domains and cell-penetrating peptides for intracellular cargo delivery.
  • Created gradients of reporter proteins (mRFP1) and a transcription factor (MyoD) to monitor transduction and gene expression.

Main Results:

  • Successfully generated stable protein transduction gradients within the 3D hydrogel matrix using GET.
  • Demonstrated spatio-temporal control of intracellular delivery of transcription factors (GET-MyoD).
  • Showed differential programming of myogenic differentiation based on location and time within the gradient.

Conclusions:

  • GET technology enables the creation of stable, diffusion-driven protein gradients in 3D hydrogels.
  • Spatio-temporal control of intracellular protein delivery can be achieved, allowing for precise programming of cell behavior.
  • This approach offers a powerful tool for regenerative medicine, facilitating tissue generation through direct gene expression programming with transcription factors.