iPSCs were generated from omental stromal cells and were a kind gift from Dr. Rivka Ofir, Ben Gurion University. The undifferentiated cells were cultivated on culture plates, pre coated with Matrigel™ (BD, Franklin Lakes, New Jersey), diluted to 250 μg/mL in DMEM/F12 (Biological Industries, Beit HaEmek, Israel). Cells were maintained in NutriStem™ (Biological Industries) medium containing 1% Penicillin/Streptomycin (Biological Industries) and cultured under a humidified atmosphere at 37°C with 5% CO2. Medium was refreshed daily and cells were passaged weekly by treatment with 1 U/mL dispase (Stemcell Technologies, Vancouver, Canada) followed by mechanical trituration.
Nutristem
Manufactured by Sartorius
Sourced in Israel
Nutristem is a cell culture medium designed for the growth and maintenance of stem cells. It provides a defined, serum-free, and animal component-free environment to support the expansion and differentiation of various stem cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Sartorius (3 mentions)
Penicillin streptomycin, by Sartorius (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Matrigel, by BD (2 mentions)
Matrigel, by Corning (2 mentions)
Dispase, by STEMCELL (1 mentions)
Matrigel, by Thermo Fisher Scientific (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Axon Medchem (1 mentions)
Relesr, by STEMCELL (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Laminin 521, by BioLamina (1 mentions)
Cytotune ips reprogramming kit, by Thermo Fisher Scientific (1 mentions)
Mtesr medium, by STEMCELL (1 mentions)
Mab4360, by Merck Group (1 mentions)
Sc5279, by Merck Group (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
M 199, by Sartorius (1 mentions)
Roswell park memorial institute (rpmi), by Bio-Techne (1 mentions)
12 protocols using nutristem
1
Expansion of Human iPSCs from Omental Stromal Cells
2
Feeder-Free Culture of Human Embryonic Stem Cells
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Human ES cell line HS415 (used from passage 17 to 30, kindly provided by Outi Hovatta, Karolinska Institute, Stockholm, Sweden) was cultured onto extracellular matrix (Matrigel, dilution 1/100, Invitrogen, Carlsbad, CA, USA) in a feeder-free culture medium (Nutristem, Biological Industries, Cromwell, CT). Medium was changed one another day to maintain pluripotency. Cells were passaged with enzymatic procedure (Accutase; Invitrogen) and replated with Rho-associated protein kinase (ROCK) inhibitor (10 μmol/l Y-27632;Axon medchem, Groningen, Netherlands) during 24 hours before removal.
3
Cultivating Human iPSCs from Omental Stromal Cells
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iPSCs were generated from omental stromal cells and were a kind gift from Dr. Rivka Ofir from Ben Gurion University. The undifferentiated cells were cultivated on 10 cm culture plates precoated with Matrigel™ (Corning, NY, USA) diluted to 250 µg·mL−1 in DMEM/F12 (Biological Industries, Kibbutz Beit-Haemek, Israel). Cells were maintained in NutriStem™ (Biological Industries) medium containing 0.1% penicillin/streptomycin (Biological Industries) and cultured under a humidified atmosphere at 37 °C with 5% CO2. The medium was refreshed daily, and cells were passaged at 70% confluence by treatment with 1 mL of ReLeSR™ (Stemcell Technologies, Vancouver, BC, Canada).
4
Characterization of Human Pluripotent Stem Cell Lines
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The VUB hESC lines were derived and characterized as previously described (Mateizel et al., 2006 (link), Mateizel et al., 2010 (link)). The lines were grown either on inactivated mouse embryonic fibroblast feeder layers using a standard hESC medium as described in (Mateizel et al., 2006 (link), Mateizel et al., 2010 (link)), or on culture dishes coated with 5 μg/mL of recombinant Laminin-521 (Biolamina) in Nutristem (Biological Industries) with 0.5% 100 U/mL penicillin/streptomycin (Thermo Fisher Scientific), as described in (Dziedzicka et al., 2016 (link)).
The VUBi004 iPSC lines were obtained by transducing fibroblasts with p4ORF-dTomato as described in Warlich et al. (2011) (link). Clonal iPSC lines were manually isolated at day 30 post-transduction, and further cultured individually on Laminin-521 and Nutristem. The iPSC lines derived in the laboratory of S. Viville, named STBG-iPSCs, were established using different reprogramming methods, as listed in theTable S1 and as described in Jung et al. (2014) (link).
The fibroblast line was established from a skin biopsy from a 53-year-old female donor. The cells were cultured in F-12 Nutrient Mix Ham (Life Technologies) with 20% fetal calf serum (Thermo Fisher Scientific), 0.5% penicillin/streptomycin (Thermo Fisher Scientific), and 1% glutamine (Thermo Fisher Scientific). The cells were expanded up to passage 4.
The VUBi004 iPSC lines were obtained by transducing fibroblasts with p4ORF-dTomato as described in Warlich et al. (2011) (link). Clonal iPSC lines were manually isolated at day 30 post-transduction, and further cultured individually on Laminin-521 and Nutristem. The iPSC lines derived in the laboratory of S. Viville, named STBG-iPSCs, were established using different reprogramming methods, as listed in the
The fibroblast line was established from a skin biopsy from a 53-year-old female donor. The cells were cultured in F-12 Nutrient Mix Ham (Life Technologies) with 20% fetal calf serum (Thermo Fisher Scientific), 0.5% penicillin/streptomycin (Thermo Fisher Scientific), and 1% glutamine (Thermo Fisher Scientific). The cells were expanded up to passage 4.
5
Generating iPSCs from Omental Stromal Cells
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iPSCs were generated
from omental stromal
cells and were a kind gift from Dr. Rivka Ofir from Ben Gurion University.
The undifferentiated cells were cultivated on 10 cm culture plates
precoated with Matrigel (BD, Franklin Lakes, New Jersey) diluted to
250 μg/mL in DMEM/F12 (Biological Industries). Cells were maintained
in a NutriStem (Biological Industries) medium containing 0.1% Penicillin/Streptomycin
(Biological Industries) and cultured under a humidified atmosphere
at 37 °C with 5% CO2. The medium was refreshed daily,
and cells were passaged at 70% confluence by treatment with 1 mL of
ReLeSR (STEMCELL Technologies, Vancouver, Canada).
from omental stromal
cells and were a kind gift from Dr. Rivka Ofir from Ben Gurion University.
The undifferentiated cells were cultivated on 10 cm culture plates
precoated with Matrigel (BD, Franklin Lakes, New Jersey) diluted to
250 μg/mL in DMEM/F12 (Biological Industries). Cells were maintained
in a NutriStem (Biological Industries) medium containing 0.1% Penicillin/Streptomycin
(Biological Industries) and cultured under a humidified atmosphere
at 37 °C with 5% CO2. The medium was refreshed daily,
and cells were passaged at 70% confluence by treatment with 1 mL of
ReLeSR (STEMCELL Technologies, Vancouver, Canada).
6
iPSC-Derived Neuron Differentiation
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iPSCs were derived from activated T cells isolated from PBMCs using a CytoTune-iPS Reprogramming Kit (Invitrogen). iPSCs were cultured on Matrigel (Corning) in mTeSR medium (Stem Cell Technologies) or Nutristem (Biological Industries). iPSCs were differentiated to neurons using the dual SMAD inhibition protocol (Chambers et al., 2009 (link)). Neuronal precursor cells derived from iPSCs were differentiated either on Matrigel or on OTBS using a previously described method (Pecho-Vrieseling et al., 2014 (link)). For detailed iPSC derivation and differentiation protocols, see Supplemental Experimental Procedures . The iPSC lines generated in this study are available upon request for research to study FXS and related diseases if all legal and ethical standards are met.
7
Maintenance of Human Pluripotent Stem Cells
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All human pluripotent stem cell studies were carried out in accordance with approval from the National University of Singapore’s Institutional Review Board (IRB 12–451). Pluripotent hESCs H1 (karyotype: 46, XY; WiCell Research Institute, WA01, RRID:CVCL_9771) were maintained on 10 μg/ml LN521 (Biolamina AB, LN521)-coated culture plates with daily change of Nutristem® (Biological Industries, 05-100-1 A) medium. Routine monitoring of pluripotent markers POU5F1 (Santa Cruz, sc-5279, RRID:AB_628051) and Tra1–60 (Millipore, MAB4360, RRID:AB_2119183) by flow cytometry and genomic stability by karyotyping were performed at the Singapore General Hospital cytogenetics laboratory. Cells were split at 200,000 cells per well and were passaged at 80% confluence by gentle dissociation with TrypLE (ThermoFisher, 12563011) at 37 °C for 8 mins to dissociate the single cells. The cell suspension was then collected and centrifuged at 800 rpm for 4 mins. Supernatants were discarded and the cell pellets were resuspended in 1 mL of pre-warm Nutristem® medium. Bright-field images were taken with a Leica microscope.
8
Cardiomyocyte Differentiation Protocol
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Prior to differentiation, cells were dissociated with Accutase™ (StemCell Technologies, Vancouver, BC, Canada) and passaged to six-well plates. Until 100% confluence of iPSCs, NutriStem™ (Biological Industries) was refreshed. On day 0, the medium was changed to 3 mL of RPMI (Biological Industries), supplemented with 0.5% L-glutamine (Biological Industries), B27-Insulin (Invitrogen, Carlsbad, CA, USA) and 4.5 µM CHIR-99021 (Tocris, Bristol, UK). On day 2, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine, B27-Insulin, and 5 µM IWP-2 (Tocris). On day 4, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine and B27-Insulin. This medium was refreshed on day 6. On day 8, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine and B27, and this medium was refreshed on day 10. From day 12, the medium was changed to M-199 (Biological Industries), supplemented with 0.1% penicillin/streptomycin, 5% fetal bovine serum (FBS, Biological Industries), 0.6 mM CuSO4·5 H2O, 0.5 mM ZnSO4·7 H2O, and 1.5 mM Vitamin B12 (Sigma-Aldrich, Darmstadt, Germany). This medium was refreshed every other day.
9
Differentiation of hES Cells into EBs
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At passage nine, after reaching confluence on LN521-coated plates, hES cells were allowed to form embryoid bodies (EBs) in 24-well ultralow attachment plates (#3473; Corning) for 14 days in NutriStem without growth factors (#06-5100-01-1A; Biological Industries). The EBs were then either plated on LN521-coated culture plates or chamber slides (#354104, Corning) for an additional 14 days. After a total of 28 days, differentiated cells were either used for gene expression analysis or fixed with 4% paraformaldehyde for immunocytofluorescence analysis.
10
Gonadal Cell Isolation and Culture
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Embryonic gonads with or without mesonephros (random allocation) were mechanically fragmented into approximately 0.5 mm3 pieces using a scalpel and enzymatically digested in Minimum essential medium alpha (MEM-α) (Gibco, 22561-021) supplemented with 1 mg/ml collagenase 1A (Sigma-Aldrich, C2674), 0.5 mg/mL DNase type 1 (Roche Diagnostics, 10104159001), and 0.5 mg/ml hyaluronidase (Sigma-Aldrich, H3506) for 15 min, at 37 °C and 120 rpm. Small cell aggregates and single cells were sedimented for 5 min at 300×g and 4 °C and resuspended in NutriStem (Biological Industries, 05-100-1A) supplemented with 1% Penicillin/Streptomycin (Gibco, 15070-063). Cell viability and concentration were determined.
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