Mem non essential amino acid

Two different types of mESC lines were used for these experiments. The first set of mESC lines—Hb9 and A2—were provided by Dr. Lee L. Rubin and were derived from either wild-type (SMN2^+/+;mSmn^+/+) or SMA (SMN2^+/+;mSmn^−/−) mice expressing the HB9:eGFP reporter construct [39] (link). The mice used to establish these mESC lines were generated by interbreeding the low-copy SMN2 carrier mouse (FVB.Cg-Tg(SMN2)89AhmbSmn^tm1Msd/J) with HB9:eGFP (mHB9:Gfp1b) transgenic mouse [37] (link), [45] (link). The second set of mESC lines—C4 and E2—were provided by Dr. Douglas Kerr [46] (link). These lines were derived from wild-type and SMA mice, respectively, and do not harbor a motor neuron-specific marker gene.
mESCs were grown as previously described [46] (link), [47] . Briefly, mESCs were grown on a primary mouse embryonic fibroblast feeder layer (Millipore, Billerica, MA, USA) in 10 cm tissue culture dishes. Cells were cultured with medium containing DMEM supplemented with 15% fetal bovine serum (Stem Cell Technologies, Vancouver, BC, Canada), 1% GlutaMax-I (Life Technologies, Carlsbad, CA, USA), 1% MEM non-essential amino acids (Millipore), 1% nucleosides (Millipore), 0.1 mM β-mercaptoethanol (Millipore), 1% penicillin/streptomycin (Life Technologies) and 10 ng/mL murine leukemia inhibitory factor (LIF; Millipore).

All media components were obtained from Life Technologies unless otherwise indicated. Dural/scalp outgrowths, established fibroblast lines, and mouse embryonic fibroblasts (MEFs, GlobalStem) were grown in fibroblast media, defined as the following: DMEM/10% FBS/Glutamax (2 mM)/2-Mercaptoethanol (0.1 mM)/Penicillin-streptomycin (100 U/mL-0.1 mg/mL). For initial plating of new dural and scalp samples, the tissue was first grown in biopsy media: DMEM/10% FBS/Glutamax (2 mM)/2-Mercaptoethanol (0.1 mM)/MEM non-essential amino acids (0.1 mM)/antibiotic-antimycotic (1×)/Nucleosides (1×, Millipore). Reprogrammed iPSCs were maintained on MEFs, in HUESM: KO-DMEM/20% KSR/Glutamax (2 mM)/2-Mercaptoethanol (0.1 mM)/bFGF (10 ng/ml)/Penicillin-streptomycin (100 U/mL-0.1 mg/mL). iPSCs were enzymatically passaged using TrypLE and replated in the presence of a Rho-kinase inhibitor (Y27632, Stemgent). Karyotyping and DNA fingerprinting of fibroblasts and iPSCs was performed by Cell Line Genetics (Madison, Wisconsin). Directed and undirected differentiations are described below.

Serum-free DMEM supplemented with 1% MEM non-essential amino acids (Millipore, Billerica, MA), 1% insulin-transferrin selenium premix (Cyagen, Guangzhou, China), 50μg/ml AA, 10^-7M dexamethasone. 5μM of 6-aminocaproic acid was added in order to prevent fibrin degradation [27 (link)]. As the bioreactor represents an open system, P/S was replaced with 100 μg/ml of Primocin (Invivogen, San Diego, CA).

Fibroblasts from index patient 1 and her parents were obtained using a punch biopsy according to standard procedures, upon informed consent (IRB approval MEC-2017-341). Fibroblasts from the parents of index patients 2 and 3 were also obtained upon informed consent at McMaster Children’s Hospital. All fibroblasts were cultured in standard DMEM medium supplemented with 15% fetal calf serum, MEM non-essential amino acids (Sigma), 100 U/ml penicillin and 100 µg/ml streptomycin, as done previously [6 ], in routine humidified cell culture incubators at 20% O₂. Fibroblast cell lines were transfected using Lipofectamine 3000 (Invitrogen) with the indicated plasmid constructs. All cell lines used in this report were regularly checked for the presence of mycoplasma and were negative during all experiments.

Neuroepithelium differentiation to primarily central nervous system epithelia requires the single-cell passaging of hESCs and the culturing of these cells to nearly 100% confluence on Matrigel-coated plates prior to beginning neural induction [19 (link), 20 (link)]. Upon reaching 100% confluence, the MEF-CM medium is replaced with N2/B27 Neural Induction medium supplemented with the small molecules SB431542 (Tocris Biosciences) and LDN193189 (Sigma). N2/B27 Neural Induction medium consists of DMEM/F12 (Gibco) as the base, N2 supplement (Invitrogen), B27 supplement minus vitamin A (Invitrogen), L-glutamine (2 mM), penicillin/streptomycin, beta-mercaptoethanol (Sigma), and MEM nonessential amino acids (Sigma). The supplemented small molecules inhibit BMP and TGF-β signalling which is paramount to successful neural induction. The medium is changed daily with a daily addition of the small molecules to final concentrations of 10 μM and 200 nM, respectively (Supplemental Figure S1). The focus for this study is the day 5 intermediate stage and the day 10 neuroepithelium. Further culturing of these cells takes place in the N2/B27 Neural Induction media without factors.

U373-MG human GBM cells (ATCC HTB-17, which have been reported to share common origins with U251-MG GBM cells) were cultured in DMEM (Invitrogen) with 10% Calf serum advantage (JR Scientific, Inc), 1% penicillin-streptomycin, 1% MEM non-essential amino acids and 1% sodium pyruvate (Sigma)26 (link). Human primary adipose derived MSCs were purchased from ATCC and maintained and grown in accordance with their recommendations. Cells were cultured and grown in MSC growth media supplemented with MSC growth supplements (ATCC) and used at passage <5. C8-B4 cells were obtained from ATCC and cultured in DMEM with 10% calf serum, 1% penicillin-streptomycin, 1% MEM non-essential amino acids and 1% sodium pyruvate. Macrophage-conditioned medium was collected after 4 days of culture. For experiments with macrophage-conditioned medium, U373-MG cells were initially seeded to patterned hydrogels in their normal growth medium and allowed to adhere to the gel overnight. The medium was then replaced with macrophage-conditioned medium and measurements were taken 2 days later.