Maintenance of Pluripotent Stem Cells

WA09 hESC (WiCell) and A04 hiPSC, a clonal derivative of HPSI0114i-kolf_2 [95 (link)], were provided by Dr. Bill Skarnes, JAX. Cells were maintained in StemFlex defined culture medium (Thermo Fisher Scientific, Waltham, MA, USA) as adherent cultures on a substrate of a recombinant fragment of human vitronectin (Synthemax, Corning, Corning, NY, USA). Cells were routinely passaged with a non-enzymatic dissociation reagent (ReLeSR, Stem Cell Technologies, Vancouver, CB, Canada). Cells were used within 15 passages from diploid master stocks, and undifferentiated cultures expressed cell surface markers and transcription factors characteristic of the pluripotent state. Bimonthly testing confirmed the absence of contamination by mycoplasma or other adventitious microbial agents. Human embryonic stem cell research was approved by the University of Connecticut Stem Cell Research Oversight Committee on behalf of JAX.

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Collin G.B., Shi L., Yu M., Akturk N., Charette J.R., Hyde L.F., Weatherly S.M., Pera M.F., Naggert J.K., Peachey N.S., Nishina P.M, & Krebs M.P. (2022). A Splicing Mutation in Slc4a5 Results in Retinal Detachment and Retinal Pigment Epithelium Dysfunction. International Journal of Molecular Sciences, 23(4), 2220.

Publication 2022

Synthemax ReLeSR

Cell Clonal Culture medium Diploid Enzymatic Hipsc Human Human embryonic stem cell Mycoplasma Stem cell Transcription factors Vitronectin

Corresponding Organization :

Other organizations : Jackson Laboratory, Cleveland Clinic, Cleveland Clinic Lerner College of Medicine, Cleveland Eye Clinic, Case Western Reserve University, Louis Stokes Cleveland VA Medical Center

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Variable analysis

independent variables

Cell type (WA09 hESC and A04 hiPSC)

dependent variables

Not explicitly mentioned

control variables

Passage number (used within 15 passages from diploid master stocks)
Cell culture conditions (maintained in StemFlex defined culture medium as adherent cultures on a substrate of a recombinant fragment of human vitronectin, routinely passaged with a non-enzymatic dissociation reagent)
Absence of contamination by mycoplasma or other adventitious microbial agents (confirmed by bimonthly testing)

controls

Positive control: Undifferentiated cultures expressing cell surface markers and transcription factors characteristic of the pluripotent state
Negative control: Not explicitly mentioned

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