Knockout serum replacement kosr

Undifferentiated hPSCs were maintained in DMEM/F-12 with 15 mM HEPES (STEMCELL Technologies), 20% KnockOut^™ serum replacement (KOSR) (Gibco), l-Glutamine (Sigma), NEAA (Life Technologies) and supplemented with 10 ng/ml FGF2 (Miltenyi Biotec) in 5% CO₂ and 100% humidity. hPSCs media were replaced every 24 h. hPSCs were manually split and seeded on irradiated CF-1 mouse embryonic fibroblasts (MTI-GlobalStem) once a week.
iAGb was cultured in feeder-free condition using TeSR™-E8™ basal medium and TeSR^™-E8^™ 25× Supplement (STEMCELL Technologies). For 17D differentiation setup, the cells were processed as according to the 17D differentiation protocol described below.
293FT cells were cultured in DMEM High glucose (Life Technologies) supplemented with 10% Fetal bovine serum (South America Hyclone) and 1% nonessential amino acid NEAA (Invitrogen).
All cell lines are screened routinely for mycoplasma contamination and are declared to be mycoplasma-free.

hESCs were cultured on irradiated mouse embryonic fibroblasts (iMEFs), which were seeded on the dishes coated with 1:6 diluted Matrigel (Corning, #354277) with hESC medium. This medium contains DMEM/F12 (Gibco, #11330-082), 20% Knockout Serum Replacement (KOSR) (Gibco, #10828028), 1 mM non-essential amino acids (Gibco, #11140-050), 1% L-glutamine (Gibco, #25-005-CI), 0.1 mM β-mercaptoethanol (Gibco, #21985023), and 10 ng ml⁻¹ bFGF (R&D System, #233-FB/CF). Cells were maintained at 37 °C with 5% CO₂ and passaged every 3–4 days by using TrypLE (Gibco, #12604013) for dissociation.

Pluripotent stem cells were routinely cultured on irradiated primary murine embryonic fibroblasts (MEF), derived from embryos of CF1 and DR4 F1 mice at embryonic days of 12.5 or 13.5 (P2/P3), or purchased from GlobalStem (Rockville, MD). Human pluripotent stem cell cultures were maintained in DMEM/F12 (Invitrogen) medium supplemented with 20% Knockout Serum Replacement (KOSR; Gibco), 0.1 mM MEM nonessential amino acids (Gibco), 1 mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and 4 ng/mL FGF2 (R&D Systems, Minneapolis, MN) at 37°C, 5% CO₂, and 85% relative humidity. The medium was changed daily on hESCs and hiPSC cultures. For experiments, human pluripotent stem cells were first transitioned from MEF feeder layers onto a BD-Matrigel™ (BD Biosciences) matrix precoated plate and cultured in mTESR1™ medium (Stem Cell Technologies, Vancouver, Canada). The mTESR1 growth media were replenished daily. Purified (>95%) human CD34⁺ CB progenitors (also referred to as “starting CB progenitors”) from pooled donors were purchased from AllCells (Emeryville, CA) and cultured in the hematopoietic growth medium (HPGM).

OG-MEFs were derived from transgenic mice at E13.5 and were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with high glucose, 1×nonessential amino acids (NEAA, Thermo), 1×L-glutamine (Thermo), β-mercaptoethanol (Gibco) and 10% fetal bovine serum (FBS). All the MEFs used for these experiments were pooled and collected before passage 3. The methods of maintaining plat-E cells and feeder cells and the viral infection methods and iPS cell induction were as previously described (1 (link)). iPS cells and mouse ES cells were maintained in knockout-DMEM medium (Gibco, N.Y, USA) containing 20% knockout serum replacement (KOSR) (Gibco, N.Y, USA), 1×Penicillin/Streptomycin Solution (P/S) (Hyclone), 1×NEAA (Thermo), 1×L-glutamine (Thermo) and β-mercaptoethanol (Gibco) with leukemia-inhibitory factor (LIF, 10 000×, Millipore). iPS cells were maintained on feeder layers of mitomycin C (Sigma)-treated MEFs.

G4 mES cells were cultured on mitomycin C mitotically inactivated MEFs as previously described [32] (link), [33] (link) using DMEM (Gibco, Carlsbad, CA)/10%FBS (HyClone, Logan, UT)/LIF at 10⁶ U/ml (Millipore, Billerica, MA)/0.1 mM b-ME (Sigma-Aldrich, St. Louis, MO) (henceforth defined as ‘mES cell media’). hES cells were cultured on gamma-irradiated MEF feeders in 80% DMEM-F12 (Gibco, Carlsbad, CA)/20% KnockOut Serum Replacement (KOSR, Gibco,)/1 mM L-glutamine (Gibco, Carlsbad, CA)/0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO)/4 ng/ml bFGF (Invitrogen, Carlsbad, CA) (henceforth defined as ‘human ES cell media’). Once ES cell cultures were sub-confluent, these cultures were either transfected with a Pax6 or Six3 expression plasmid or infected with the appropriate lentiviral vectors. Pax6 and Six3 expression plasmid vectors were transfected into G4 or Pax6-GFP mES cells using FuGENE 6 (Roche, Madison, WI). Pax6 and Six3 lentiviral vectors were introduced by infecting cells with freshly harvested viral supernatant along with 6 µg/ml of polybrene (Millipore, Billerica, MA). Transfected and transduced ES cells cultures were fixed on days 7, 14, 21, 28 or 30 in vitro using 4% paraformaldehyde/4% sucrose at room temperature for 15 minutes. The cultures were then processed for immunostaining as described below.

hiPSC colonies were treated with collagenase IV (GIBCO BRL Life Technologies, Invitrogen Life Technologies, Carlsbad, CA, USA) and gently scraped off the culture dishes. After centrifugation, cells were resuspended in the EB medium (DMEM/F12 supplemented with 20% KnockOut™ Serum Replacement (KOSR, GIBCO BRL Life Technologies, Invitrogen Life Technologies, Carlsbad, CA, USA), 2 mM Glutamax, 1% MEM non-essential amino acids (MEM-NEAA), and 55 µM 2-mercaptoethanol) and transferred into low attachment dishes. EBs were cultured in a 37 °C incubator with humidified atmosphere of 5% CO₂. The medium was changed every other day. After 14 days EBs were collected and assessed by RT-PCR for expression of stem cell and differentiation markers.