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. 2019 Feb 14;9:2072. doi: 10.1038/s41598-018-37430-9

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Generation of iPS cell lines doxycycline-inducible overexpressing LHX2 (iLHX2) and differentiation of iLHX2 into iPS-HSCs. (a) Schema for construct of a self-contained, drug-inducible expression vector based on the PiggyBac transposon (PB-T-LHX2-G-ERN) used in this study. Tetracycline resistant operon (tetO), LHX2 gene, internal ribosomal entry site (IRES), green fluorescence protein (GFP), EF1α promoter sequences (EF1α), reverse tetracycline transactivator gene (rtTA), and neomycin-resistant gene (neo) are shown. (b) Representative view of pooled iPS cell clones containing genomic transposon integrations of LHX2 (iLHX2-iPS). Activation of LHX2 expression in response to Dox was indirectly monitored by co-incident GFP (green). Scale bars: 200 μm. (c) Expression of OCT3/4 and LHX2 in iLHX2-iPS clones cultured with vehicle (Veh) or doxycycline (Dox). The y-axis represents the ratio of expression relative to the means of iPS cells. (d) Immunoblot analysis of iLHX2-iPS clones and iPS-HSCs derived from iLHX2-iPS clones (iLHX2-HSC), which were cultured with Veh or Dox. (e) Expression of PDGFRα, NGFR, CYGB, and HGF in iLHX2-iPS clones, iPS-MP derived from iLHX2-iPS clones (iLHX2-MP), and iLHX2-HSCs, which were cultured without Dox. (f) Left panel: protocol for iLHX2-HSCs cultured with Dox at days 4–6. Expression of LHX2, NGFR, and CYGB in iLHX2-HSCs cultured with Veh or Dox. Results represent the mean ± SD of three separate experiments. *P < 0.05.