Expansion and Maintenance of HUDEP2 and Tet-On-Cas9 iPSCs

HUDEP2 (HUDEP-2) cells (a kind gift from Yukio Nakamura) (Kurita et al., 2013 (link)) were cultured following previously reported protocols (Kurita et al., 2013 (link); Bagchi et al., 2021 (link)). Briefly, the cells were expanded in StemSpan SFEM-II medium (Stem Cell Technologies) supplemented with 1 µM dexamethasone, 1 μg/mL doxycycline (Dox), 50 ng/mL recombinant human stem cell factor (rh SCF), 3 units/mL recombinant human erythropoietin (rh EPO), 10 ng/mL recombinant human interleukin 3 (rh IL-3) and 1% penicillin/streptomycin. The AAVS1-Tet-On-Cas9 iPSCs generated in our laboratory (Thamodaran et al., 2022 (link)) were cultured in Matrigel (Corning) - coated plates containing mTeSR medium (STEMCELL Technologies). Upon reaching 70%–80% confluency, colonies were dissociated with Versene (Thermo Fisher Scientific) following the manufacturer’s protocol and passaged at a 1:4 ratio.

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Ijee S., Chambayil K., Chaudhury A.D., Bagchi A., Modak K., Das S., Benjamin E.S., Rani S., Paul D.Z., Nath A., Roy D., Palani D., Priyanka S., Ravichandran R., Kumary B.K., Sivamani Y., S. V., Babu D., Nakamura Y., Thamodaran V., Balasubramanian P, & Velayudhan S.R. (2024). Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs. Frontiers in Molecular Biosciences, 10, 1295507.

Publication 2023

StemSpan SFEM-II medium Matrigel mTeSR medium Versene

Corresponding Organization :

Other organizations : Sree Chitra Thirunal Institute for Medical Sciences and Technology, Regional Centre for Biotechnology, Manipal Academy of Higher Education, Tata Institute for Genetics and Society

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Variable analysis

independent variables

Dexamethasone concentration (1 µM)
Doxycycline concentration (1 μg/mL)
Recombinant human stem cell factor (rh SCF) concentration (50 ng/mL)
Recombinant human erythropoietin (rh EPO) concentration (3 units/mL)
Recombinant human interleukin 3 (rh IL-3) concentration (10 ng/mL)

dependent variables

Not explicitly mentioned

control variables

Cell culture medium (StemSpan SFEM-II)
Penicillin/streptomycin concentration (1%)
Cell line (HUDEP2 cells)
Matrigel-coated plates
MTeSR medium (for iPSCs)

positive controls

Not mentioned

negative controls

Not mentioned

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