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Optimization of Differentiation Media for Human Induced Pluripotent Stem Cells using Design of Experiments
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  • Rana Khalife ,
  • Patricia Esteban,
  • Hamza Patel,
  • Farlan Veraitch
Rana Khalife
UCL
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Patricia Esteban
Aston University
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Hamza Patel
UCL
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Farlan Veraitch
UCL
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Abstract

The implementation of human pluripotent stem cells (hiPSCs) in cell therapy has an extraordinary potential but faces many practical challenges, including costs associated with growth media and factors. There is an immediate need to establish an optimized culture platform to direct the differentiation of hiPSCs into germ layers in a defined nutritional microenvironment to generate cost-effective and robust therapeutics. The aim of this study was to identify the optimal nutritional environment by mimicking the in vivo concentrations of three key factors (glucose, pyruvate and oxygen) during the spontaneous differentiation of hiPSCs derived from cord blood, which greatly differ from the in vitro expansion and differentiation scenarios. Moreover, we hypothesized that the high glucose, pyruvate and oxygen concentrations found in typical growth media could inhibit the differentiation of certain lineages. A design of experiments (DoE) was used to investigate the interaction between these three variables during the spontaneous differentiation of hiPSCs. We found that lower oxygen and glucose concentrations enhance the expression of mesodermal (Brachyury, KIF1A) and ectodermal (Nestin, B-Tubulin) markers. Our findings present a novel approach for efficient directed differentiation of hiPSCs through the manipulation of media components whilst simultaneously avoiding the usage of growth factors thus reducing costs.

Peer review status:IN REVISION

23 Oct 2020Submitted to Biotechnology Journal
31 Oct 2020Assigned to Editor
31 Oct 2020Submission Checks Completed
31 Oct 2020Reviewer(s) Assigned
16 Nov 2020Editorial Decision: Revise Major