Knockout serum replacement ksr

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Knockout Serum Replacement (KSR) is a cell culture supplement designed to support the growth and maintenance of embryonic stem cells and induced pluripotent stem cells. It is a chemically defined, animal-component-free alternative to fetal bovine serum.

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84 protocols using knockout serum replacement ksr

Maintenance and Passaging of hESC X-01

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The hESC cell line X-01 [15 (link)], provided by the Stem Cell Bank, Chinese Academy of Sciences, was cultured on irradiated inactivated CF-1 MEF in DMEM/F12 supplemented with 20% Knockout Serum Replacement (KSR, Invitrogen), 200 mM glutamine, 55 mM 2-mercaptoethanol, 1% nonessential amino acids (all from Invitrogen), and 8 ng/mL FGF-2 (Peprotech), for long term maintenance. hESCs were cultured on methanol fixed xeno-free ECM plate with either mTeSR1 (STEMCELL) [16 (link)] or TeSR-E8 medium (STEMCELL) [17 (link)].
hESCs grown on methanol fixed xeno-free ECM were passed by Dispase (1 mg/mL) for 5 minutes at 37°C or by Gentle Cell Dissociation Reagent (STEMCELL) for 5 minutes at room temperature, followed by cell detachment with a scraper.

Zhu H., Yang J., Wei Y, & Chen H.H. (2015). Development of a Xeno-Free Substrate for Human Embryonic Stem Cell Growth. Stem Cells International, 2015, 621057.

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Rhesus Embryonic and Induced Pluripotent Stem Cells

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WT Rhesus ESCs and transgenic Rhesus HD iPSCs were previously established [90 (link)]. Transgenic HD iPSCs expressed both exon 1 of the human HTT gene with 65 CAG repeats and GFP under the control of the human polyubiquitin-C (UBC) promoter [88 (link), 90 (link)]. Rhesus PSCs were cultured on mouse fetal fibroblast (MFF) feeder cells in ESC culture media [Knockout-Dulbecco’s modified Eagle’s medium (KO-DMEM; Invitrogen) with 20% Knockout Serum Replacement (KSR; Invitrogen), 1 mM glutamine, 1 nonessential amino acids (NEAA), and 4 ng/mL of human basic fibroblast growth factor (bFGF; Chemicon, Inc., Tumecula, CA)]. In addition, Rhesus PSC cultures were expanded by mechanical passaging. Since these Rhesus PSC lines were previously used to derive stable NPC cultures [89 (link), 137 (link), 138 (link)], PSCs were collected for experimental analysis, but not further differentiated into NPCs in this study.

Goodnight A.V., Kremsky I., Khampang S., Jung Y.H., Billingsley J.M., Bosinger S.E., Corces V.G, & Chan A.W. (2019). Chromatin accessibility and transcription dynamics during in vitro astrocyte differentiation of Huntington’s Disease Monkey pluripotent stem cells. Epigenetics & Chromatin, 12, 67.

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Serum-free culture medium optimization

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Preliminary experiments were performed according to previously published protocols, mostly using fetal calf serum based medium, supplemented with growth factors (i.e., Ootani et. al., 2009 (link)). To increase experiments reproducibility and support standardization, we developed a serum-free culture medium, based on the KnockOut serum replacement (KSR, Invitrogen) and supplements to support the different cellular populations in the tissue (stem cells, neurons, immune cells, etc… See above full description of medium composition). Additional growth factors tested during the optimization phase, but were not included in the final medium formulation, include EGF (50–500ng/ml), R-spondin1 (500ng–5ug/ml), Amphiregulin (5ug/ml). We also tested AlboMAX (Invitrogen), Prostaglandin E2, Nicotinamide, Dexamethasone, Sodium pyruvate and beta-mercaptoethanol. These factors were eventually not used in this study, as we did not observe any visible change to overall tissue structure or viability.

Yissachar N., Zhou Y., Ung L., Lai N.Y., Mohan J.F., Ehrlicher A., Weitz D.A., Kasper D.L., Chiu I.M., Mathis D, & Benoist C. (2017). An intestinal organ culture system uncovers a role for the nervous system in microbe-immune crosstalk. Cell, 168(6), 1135-1148.e12.

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Differentiation of Mouse ESCs into Cortical Neurons

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Bcl11b-IRES-EGFP knock-in mouse ESCs were maintained and cultured as previously reported (Watanabe et al., 2005 (link)). The differentiation medium was constituted of G-MEM supplemented with 10% Knockout Serum Replacement (KSR; Invitrogen), 2 mM L-glutamine (Thermo Fisher Scientific), 1 mM Sodium pyruvate solution (SIGMA), 0.1 mM MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific), 0.1 mM 2-Mercaptoethanol (Wako, Japan), 10 μM SB431542 (Merck), and 20 nM Wnt-C59 (Cellagen Technology) (Eiraku et al., 2008 (link); Motono et al., 2016 (link)). For the SFEBq culture, ESCs were dissociated into single cells in 0.05% trypsin-EDTA (Invitrogen) and quickly reaggregated in the differentiation medium (4000 cells/150 μl/well) using Prime Surface 96U plates (Sumilon). To induce mouse ESC-derived cortical neurons, day 6 cell aggregates were transferred to a 10 cm bacterial-grade dish in N2 medium (DMEM/F12 supplemented with N2, B27, 0.1 mM 2-ME, and 2 mM glutamine) supplemented with 50 ng/ml FGF8b (R&D systems) and 5 μM cyclopamine (Enzo life sciences) for dorso-anteriorization of the telencephalon.

Sunohara T., Morizane A., Matsuura S., Miyamoto S., Saito H, & Takahashi J. (2019). MicroRNA-Based Separation of Cortico-Fugal Projection Neuron-Like Cells Derived From Embryonic Stem Cells. Frontiers in Neuroscience, 13, 1141.

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Porcine Induced Pluripotent Stem Cells

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Porcine ear fibroblasts (PEFs) were derived from a 10-day-old miniature pigs.
Lentiviral transduction was performed using the viPS Vector Kit (Thermo Fisher
Scientific) following the manufacturer’s instructions. The PEFs were then
transduced with lentiviral vectors encoding six human transcription factors
(OCT4, NANOG, SOX2, C-MYC, KLF4, and
LIN28) as previously described. Established piPSCs were
cultured on mitomycin C inactivated mouse embryonic fibroblasts (MEF) feeder
with stem cell medium (DMEM/F12 culture medium supplemented with 10% Knockout
serum replacement (KSR; Invitrogen), 10% FBS (Invitrogen), 50 units/mL
penicillin (GIBCO), 50 μg/mL streptomycin (GIBCO), 2 mM L-glutamine (GIBCO), 0.1
mM nonessential amino acids (NEAAs, GIBCO), 1 μM β-mercaptoethanol and 20 ng/mL
leukemia inhibitory factor (LIF; Sigma). Colonies were passaged manually into 35
mm MEF culture dishes with daily exchange of fresh stem cell medium and
maintained by manual passage every 4-5 days. Invitro differentiation was determined by embryoid body (EB)
formation. EBs were produced using the Aggrewell plate (Stemcell Technologies)
following the manufacturer’s instructions. The aggregated cells were then
transferred to a Petri-dish (BD Falcon) suspension culture in stem cell medium
without LIF and the medium was changed every other day for 10 days.

Kwon D.J., Hwang I.S., Kwak T.U., Yang H., Park M.R., Ock S.A., Oh K.B., Woo J.S., Im G.S, & Hwang S. (2017). Effects of Cell Cycle Regulators on the Cell Cycle Synchronization of Porcine induced Pluripotent Stem Cells. Development & Reproduction, 21(1), 47-54.

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Rat iPSC Culture and Characterization

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The rat iPSCs line used in this study was provided by Dr. Lei Xiao[25 (link)]. T25 Corning flasks were incubated with 0.1% gelatin for 30 min. at 37°C, then murine embryonic fibroblasts (MEFs, 5×10⁴ cells/cm²) were cultured in fibroblasts medium (Dulbecco's modified Eagle's medium, DMEM) containing 10% fetal bovine serum (FBS), and 100 IU/ml penicillin/streptomycin (all from Invitrogen except FBS from Hyclone) two days before plating rat iPSCs. iPSCs were washed and centrifuged (1000 rpm×5min.) in a 15 ml centrifuge tube containing 5 ml iPSCs culture medium twice and then plated on confluent MEF feeder in iPSCs culture medium containing Knockout DMEM supplemented with 10% Knockout Serum Replacement (KSR), 1 mM glutamine, 1% NEAAs, and 0.1 mM β-mercaptoethanol (all from Invitrogen). After two days of culture, the iPSCs formed clones on MEFs. Then the iPSCs were passaged in the medium every 3 to 5 days. Medium was changed every other day. Alkaline phosphatase (AP) staining was conducted to identify these iPSCs clones as previously described [13 (link)].

Qin J., Ma X., Qi H., Song B., Wang Y., Wen X., Wang Q.M., Sun S., Li Y., Zhang R., Liu X., Hou H., Gong G, & Xu Y. (2015). Transplantation of Induced Pluripotent Stem Cells Alleviates Cerebral Inflammation and Neural Damage in Hemorrhagic Stroke. PLoS ONE, 10(6), e0129881.

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Isolation and Culture of Pituitary Adenoma and Meningioma Cells

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Before isolating primary pituitary adenoma and meningioma cells, all pituitary adenoma and meningioma biopsies were washed in PBS supplemented with P/S and 10% FBS in order to remove unhealthy tissues. After carefully washing, the remaining healthy tissues were cut into small pieces using refined scissors and dissociated into small cell clumps (10–50 cells/clump) with pre-chilled accutase. Then these small clumps of primary pituitary adenoma and meningioma cells were collected and re-suspended in cell culture medium: DMEM/F12 (Invitrogen), 1% Knock Out serum replacement (KSR, Invitrogen), N2 supplement (100X, Invitrogen), B27 supplement (100X, Invitrogen),1 mM l-Glutamine, 0.1 mM NEAA, 0.1 mM 2-ME with 4 ng/ml bFGF (R&D systems), and 10 ng/ml EGF (R&D systems). After evaluating the cell number and viability, primary pituitary adenoma and meningioma cells were seeded at 2 × 10⁵ cells/well (6 well plate) on Matrigel-embedded plate. The medium was replenished every 24 h for 5–7 days before passaging. Pituitary adenoma and meningioma cells can be passaged by accutase digestion and can be routinely maintained in cell culture medium for 5–10 passages.

Zhu H., Miao Y., Shen Y., Guo J., Xie W., Zhao S., Dong W., Zhang Y, & Li C. (2019). The clinical characteristics and molecular mechanism of pituitary adenoma associated with meningioma. Journal of Translational Medicine, 17, 354.

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Maintenance of Human Pluripotent Stem Cells

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Human ESCs (khES-2 strain obtained from Kyoto University) were maintained in an undifferentiated state as described previously ^{(16) (link)}. Briefly, human ESCs were co-cultured with mouse embryonic fibroblasts (MEFs) in human ESC medium composed of 80% D-MEM/Ham’s F-12 medium (1/1 ratio; Wako Pure Chemical Industries, Osaka, Japan), 20% Knockout Serum Replacement (KSR; Invitrogen, Carlsbad, CA, USA), 0.1 mM nonessential amino acids (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine (Sigma-Aldrich), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), and 4 ng/mL basic fibroblast growth factor (Wako).
Human iPSCs (253G1 strain obtained from Center for iPSC Research and Application, Kyoto University) were maintained under feeder-free conditions in Essential 8^® medium (Invitrogen) on culture dishes coated with 0.5 μg/cm² iMatrix-511 (Nippi, Tokyo, Japan).

Yamashita H., Fukuda K, & Hattori F. (2019). Hepatocyte-like Cells Derived from Human Pluripotent Stem Cells Can Be Enriched by a Combination of Mitochondrial Content and Activated Leukocyte Cell Adhesion Molecule. JMA Journal, 2(2), 174-183.

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Culturing Human Induced Pluripotent Stem Cells

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The human iPSC line iPS-201B-7 was purchased from RIKEN BRC. Additional cell lines, hi25-4 (derived from human cord blood), hi76-2 (derived from T cells), hi6 (derived from fibroblast) were kindly distributed by Dr.Koseki's laboratory in RIKEN Center for Integrative Medical Sciences.
Cells were cultured on mitomycin C (Sigma Aldrich) treated SNL76/7 cells (ECACC, Porton Down, UK) in the Primate pluripotent stem cells media from ReproCell (RCHEMD 001) containing 4 ng/ml basic Fibro Growth Factor (bFGF). The cells were passaged in small clumps after treatment to remove feeder cells with CTK solution, containing 0.025% trypsin, 0.1 mM collagenase IV, 0.1 mM CaCl2 and 20% Knockout serum replacement (KSR) (Invitrogen, Carlsbad, CA) every 3–5 days. Recombinant CCL2 (WAKO, Osaka, Japan) was added into culture medium with concentration of 10 ng/ml for culturing of human iPS cells. CCL2 was added into the medium everyday when the medium was changed.

Hasegawa Y., Tang D., Takahashi N., Hayashizaki Y., Forrest A.R., the FANTOM c.o.n.s.o.r.t.i.u.m, & Suzuki H. (2014). CCL2 enhances pluripotency of human induced pluripotent stem cells by activating hypoxia related genes. Scientific Reports, 4, 5228.

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Fibroblast Reprogramming into iPSCs

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Fibroblasts were reprogrammed into iPSCs using standard techniques as described previously (Guo et al. 2013). In brief, VSV‐g pseudo‐typed lentivirus containing a polycistronic coding sequence for the human genes OCT3/4, SOX2, KLF4, and MYC (hSTEMCCA vector kindly provided by Dr. Gustavo Mostoslavsky, Boston University School of Medicine) was used to infect fibroblasts. Seven days after infection, fibroblasts were transferred to a cell feeder layer of mouse embryonic fibroblasts and fed every other day with human pluripotent stem cell media consisting of DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 20% knockout serum replacement (KSR; Invitrogen), 2 mM glutamax (Invitrogen), 1× nonessential amino acids (Invitrogen), and 10 ng/mL FGF2. At day 21 after infection, individual iPSC clones were manually picked and dissociated for expansion into iPSC lines.

Sheng Y., Filichia E., Shick E., Preston K.L., Phillips K.A., Cooperman L., Lin Z., Tesar P., Hoffer B, & Luo Y. (2016). Using iPSC‐derived human DA neurons from opioid‐dependent subjects to study dopamine dynamics. Brain and Behavior, 6(8), e00491.

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