title: Functionalizing Ascl1 with Novel Intracellular Protein Delivery Technology for Promoting Neuronal Differentiation of Human Induced Pluripotent Stem Cells.
abstract: Pluripotent stem cells can become any cell type found in the body. Accordingly, one of the major challenges when working with pluripotent stem cells is producing a highly homogenous population of differentiated cells, which can then be used for downstream applications such as cell therapies or drug screening. The transcription factor Ascl1 plays a key role in neural development and previous work has shown that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly into neurons. Here we report on how a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. We first evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media. Next, we looked at the frequency of Ascl1-IPTD supplementation in the media on differentiation and found that one time supplementation is sufficient enough to trigger the neural differentiation process. Ascl1-IPTD was efficiently taken up by the hiPSCs and enabled rapid differentiation into TUJ1-positive and NeuN-positive populations with neuronal morphology after 8 days. After 12 days of culture, hiPSC-derived neurons produced by Ascl1-IPTD treatment exhibited greater neurite length and higher numbers of branch points compared to neurons derived using a standard neural progenitor differentiation protocol. This work validates Ascl1-IPTD as a powerful tool for engineering neural tissue from pluripotent stem cells.
authors.affiliation: Biomedical Engineering Program, University of Victoria, Victoria, BC, Canada.
authors.forename: Meghan
authors.initials: M
authors.lastname: Robinson
authors.affiliation: Department of Biochemistry, University of Victoria, Victoria, BC, Canada.
authors.forename: Parv
authors.initials: P
authors.lastname: Chapani
authors.affiliation: Biomedical Engineering Program, University of Victoria, Victoria, BC, Canada.
authors.forename: Tara
authors.initials: T
authors.lastname: Styan
authors.affiliation: iProgen Biotech, Inc., Vancouver, BC, Canada.
authors.forename: Ranjani
authors.initials: R
authors.lastname: Vaidyanathan
authors.affiliation: Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada. willerth@uvic.ca.|Division of Medical Sciences, University of Victoria, Victoria, BC, Canada. willerth@uvic.ca.|International Collaboration on Repair Discoveries (ICORD), Vancouver, BC, Canada. willerth@uvic.ca.
authors.forename: Stephanie Michelle
authors.initials: SM
authors.lastname: Willerth
mesh_terms: D034961:Antigens, Nuclear / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D018014:Gene Transfer Techniques; D006801:Humans; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism
chemical_list: D034961:Antigens, Nuclear; D051792:Basic Helix-Loop-Helix Proteins; D016895:Culture Media, Serum-Free; D009419:Nerve Tissue Proteins; D014404:Tubulin; C065500:ASCL1 protein, human; C498585:TUBB3 protein, human; C506808:neuronal nuclear antigen NeuN, human
keywords: Drug delivery; Intracellular targeting; Neurite extension; Neuroscience; Proteins; Stem cells