Systematic optimization of human pluripotent stem cells media using Design of Experiments.
Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin-1 beta 1 (NRG1β1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.
University of California San Diego, School of Medicine, Department of Pediatrics/Rady Children's Hospital San Diego, Department of Cellular &Molecular Medicine, Stem Cell Program, La Jolla, CA 92093, MC 0695, USA.
Paulo A
PA
Marinho
University of California San Diego, School of Medicine, Department of Pediatrics/Rady Children's Hospital San Diego, Department of Cellular &Molecular Medicine, Stem Cell Program, La Jolla, CA 92093, MC 0695, USA.
Thanathom
T
Chailangkarn
University of California San Diego, School of Medicine, Department of Pediatrics/Rady Children's Hospital San Diego, Department of Cellular &Molecular Medicine, Stem Cell Program, La Jolla, CA 92093, MC 0695, USA.
Alysson R
AR
Muotri
D000465:Algorithms; D061249:Batch Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation / Q000502:physiology; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015233:Models, Statistical; D020890:Neuregulin-1 / Q000737:chemistry / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology*; D012107:Research Design
D017077:Culture Media, Conditioned; C094131:NRG1 protein, human; D020890:Neuregulin-1; D016222:Fibroblast Growth Factor 2