Abstract
Advances in synthetic biology have enabled robust control of cell behavior by using tunable genetic circuits to regulate gene expression in a ligand-dependent manner. Such circuits can be used to direct the differentiation of pluripotent stem cells (PSCs) towards desired cell types, but rational design of synthetic gene circuits in PSCs is challenging due to the variable intracellular environment. Here, we provide a framework for implementing synthetic gene switches in PSCs based on combinations of tunable transcriptional, structural, and posttranslational elements that can be engineered as required, using the vanillic acid-controlled transcriptional activator (VanA) as a model system. We further show that the VanA system can be multiplexed with the well-established reverse tetracycline-controlled transcriptional activator (rtTA) system to enable independent control of the expression of different transcription factors in human induced PSCs in order to enhance lineage specification towards early pancreatic progenitors. This work represents a first step towards standardizing the design and construction of synthetic gene switches for building robust gene-regulatory networks to guide stem cell differentiation towards a desired cell fate.
Highlights
IntroductionHuman pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are unspe cialized cells defined by their ability to self-renew and to differentiate in vitro into the three germ layers (ectoderm, mesoderm, and endoderm) (Itskovitz-Eldor et al, 2000)
Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, are unspe cialized cells defined by their ability to self-renew and to differentiate in vitro into the three germ layers (Itskovitz-Eldor et al, 2000)
In order to characterize the performance of synthetic genes switches and how they can differ between immortalized cell lines and Human pluripotent stem cells (hPSCs), we selected three previously described synthetic transcription activation systems, the erythromycin-responsive (E-VP16; ET1) system derived from Escherichia coli (Weber et al, 2002), the phloretin-responsive (TtgR-VP16; TtgA1) system derived from Pseudomonas putida (Git zinger et al, 2009), and the vanillic acid-responsive (VanR-VP16; vanillic acid-controlled transcriptional activator (VanA) 1) system derived from Caulobacter crescentus (Gitzinger et al, 2012)
Summary
Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are unspe cialized cells defined by their ability to self-renew and to differentiate in vitro into the three germ layers (ectoderm, mesoderm, and endoderm) (Itskovitz-Eldor et al, 2000). The challenge in engineering stem cells is to develop advanced strategies to mimic the developmental niches that guide cell fate by exposing differentiating cells to a controlled environment using exogenous signals (Peerani and Zandstra, 2010). To this end, significant effort has been dedicated to identify the factors and optimal conditions needed to control lineage commitment (Loh et al, 2014, 2016; Przybyla et al, 2016; Tchieu et al, 2017). To fully realize the potential of hPSCs, we need to devise better differen tiation strategies with reduced variability, complexity and cost compared with current protocols
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