Spinal cords from adult Naive, Sham, and 7 dpi mice were dissected and dissociated as described above. FACS-sorted tdT + cells were directly plated in poly-d-lysin and laminin coated well chamber slides in the differentiation conditions. Astrocyte differentiation media contained DMEM/F-12 media supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; cat #16000044) and 1% P/S. Neuronal differentiation media contained DMEM/F-12 media supplemented with 5 μg/mL Heparin, N2, 2% B27, 20 ng/mL NT3 (Stemcell Technologies; cat #78074), 20 ng/mL brain-derived neurotrophic factor (BDNF; Stemcell Technologies; cat. #78005) and 1% P/S. Oligodendrocyte differentiation media contained (DMEM)/F-12 media supplemented with 5 μg/mL Heparin, N2, B27, 20 ng/mL 3,3′,5-Triiodo-L-thyronine (T3; Sigma-Aldrich; cat #T6397), 20 ng/mL human recombinant platelet-derived growth factor (PDGF-AA; Stemcell Technologies; cat #78095), 20 ng/mL NT3 and 1% P/S. Cells were maintained at 37°C with 5% CO2 in a humidified incubator with media changed every 2–3 days. Cells were fixed with 4% PFA for 20 min between 7 and 14 days after plating and then stained with the immunocytochemistry protocol described above.
Pdgf aa
Manufactured by STEMCELL
Sourced in Canada, United States
PDGF-AA is a recombinant human platelet-derived growth factor, specifically the AA homodimer. It is a key regulator of cell growth, cell proliferation, and cell differentiation, particularly in mesenchymal cells.
Automatically generated - may contain errors
Lab products found in correlation
Heparin, by STEMCELL (6 mentions)
Neurobasal medium, by Thermo Fisher Scientific (4 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (4 mentions)
Human egf, by STEMCELL (4 mentions)
Human basic fgf, by STEMCELL (4 mentions)
Pdgf bb, by STEMCELL (4 mentions)
Hepes buffer, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Heparin, by Merck Group (2 mentions)
Brain derived neurotrophic factor (bdnf), by STEMCELL (2 mentions)
3 3 5 triiodo l thyronine t3, by Merck Group (2 mentions)
Normal healthy astrocytes nhas, by Lonza (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by STEMCELL (1 mentions)
N2 supplement, by STEMCELL (1 mentions)
7 protocols using pdgf aa
1
Differentiation of Neural Cell Types
2
DIPG Cell Culture Protocol
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Primary DIPG lines were grown as previously reported in stem cell media containing half quantity of DMEM/F12 and half of Neurobasal medium (Invitrogen) supplemented with Glutamax, Pyruvate, Non-essential amino acids, Hepes buffer, antibiotic/antimycotic (Invitrogen), heparin (Stem Cell technologies), human EGF, human basic FGF, PDGF-AA, PDGF-BB (Stem Cell Technologies). Normal Healthy Astrocytes (NHAs) (Lonza) were grown according to the manufacturer’s instructions. All cells were maintained in a humidified atmosphere at 37° C and 5% CO2.
3
DIPG Cell Culture Protocols
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Primary DIPG cultures were grown mainly as neurospheres in all the experiments listed in this study. All cultures have been validated by STR profiling and mycoplasma testing. DIPG cells were use within the following passage ranges HSJD-DIPG007—p36–37, SU-DIPGVI—p29-p41, RA055—P2-P5 and VUMC-DIPG10—P3-P6. DIPG cultures were grown in stem cell media containing half quantity of DMEM/F12 and half of the Neurobasal medium (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) supplemented with glutamax, pyruvate, non-essential amino acids, HEPES buffer, antibiotic/antimycotic (Invitrogen). Stem cell media were freshly supplemented each time with heparin (Stem Cell Technologies, Vancouver, BC, Canada), human EGF, human basic FGF, PDGF-AA and PDGF-BB (Stem Cell Technologies) [44 (link)]. Cultures were maintained at 37° C in a humidified atmosphere with 5% CO2.
4
Culturing Patient-Derived DIPG and Thalamic Cells
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Patient-derived DIPG and thalamic cells were grown in cancer stem cell (CSC) media consisting of a 50:50 mixture of DMEM/F12 and Neurobasal medium (Invitrogen) supplemented with heparin (Stem Cell Technologies), glutamax, pyruvate, non-essential amino acids, Hepes buffer and antibiotic/antimycotic (Invitrogen). In order to grow the primary cells as neurospheres, CSC media was also supplemented with growth factors such as human EGF, human basic FGF, PDGF-AA, and PDGF-BB (Stem Cell Technologies). Human healthy lung fibroblasts (MRC5) (ATCC) and normal healthy astrocytes (NHAs) (Lonza) were cultured according to the manufacturer’s instructions. RA038 and P000302 cultures were developed from ZCC/PRISM clinical trial as described in7 (link) and grown in CSC media with the addition of 5% fetal calf serum. DIPG and normal cells were cultured in T75 cm2 flasks at 37 °C in a humidified atmosphere with 5% CO2. ZCC/PRISM clinical trial is approved by the Hunter New England Human Research Ethics Committee. Written informed consent was received from all participants. Specific information on tissue type each culture is derived from, in mutational status, as well as patient treatment information is included in Supplementary Table 5 .
5
Culturing Tumor Cells from Spinal Cord
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After sacrificed the minipigs, the tumors were collected from the spinal cord of the animal. Core and edge cell samples were cultured in Dulbecco's Modified Eagle Medium (DMEM), with addition of media components including 10 ng/mL PDGFAA (# 78,095, STEMCELL Technologies, USA),10 ng/mL bFGF (# 78,003.1, STEMCELL Technologies, USA), N2 Supplement (07152, STEMCELL Technologies, USA), 20% FBS and penicillin (100units/mL)-streptomycin (100 μg/mL) (Hyclone, USA) in a humidified incubator with 5% CO2 at 37 °C according to a previously described protocol [34 ].
6
Differentiation of Neural Cell Types
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Spinal cords from adult Naive, Sham, and 7 dpi mice were dissected and dissociated as described above. FACS-sorted tdT + cells were directly plated in poly-d-lysin and laminin coated well chamber slides in the differentiation conditions. Astrocyte differentiation media contained DMEM/F-12 media supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; cat #16000044) and 1% P/S. Neuronal differentiation media contained DMEM/F-12 media supplemented with 5 μg/mL Heparin, N2, 2% B27, 20 ng/mL NT3 (Stemcell Technologies; cat #78074), 20 ng/mL brain-derived neurotrophic factor (BDNF; Stemcell Technologies; cat. #78005) and 1% P/S. Oligodendrocyte differentiation media contained (DMEM)/F-12 media supplemented with 5 μg/mL Heparin, N2, B27, 20 ng/mL 3,3′,5-Triiodo-L-thyronine (T3; Sigma-Aldrich; cat #T6397), 20 ng/mL human recombinant platelet-derived growth factor (PDGF-AA; Stemcell Technologies; cat #78095), 20 ng/mL NT3 and 1% P/S. Cells were maintained at 37°C with 5% CO2 in a humidified incubator with media changed every 2–3 days. Cells were fixed with 4% PFA for 20 min between 7 and 14 days after plating and then stained with the immunocytochemistry protocol described above.
7
Primary brain tumor culture protocol
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Primary brain tumor cultures were grown in stem cell media containing DMEM/F12 and Neurobasal medium (Invitrogen, Waltham, MA, USA) (50:50) supplemented with glutamax, pyruvate, non-essential amino acids, HEPES buffer, and antibiotic/antimycotic (Invitrogen). During culture passage, stem cell media were freshly supplemented each time with heparin (Stem Cell Technologies, Tullamarine, Australia), human EGF, and human basic FGF (Stem Cell Technologies). DIPG cultures were also supplemented with PDGF-AA and PDGF-BB (Stem Cell Technologies). In some cultures, 5% of fetal calf serum was added. Cultures were maintained at 37 • C in a humidified atmosphere with 5% CO 2 . HSJD-DIPG007 and HSJD-GBM2 were kindly provided by Dr. A. Montero-Carcaboso (St John of Hope Hospital, Barcelona, Spain), whereas SU-DIPG cultures and VUMC-DIPG10 were generously given by Prof M. Monje (Stanford University, Stanford, CA, USA) and Prof E. Hulleman (Amsterdam University Medical Center, Amsterdam, The Netherlands), respectively. The remaining cultures were established as part of the Zero Childhood Cancer/PRISM personalized medicine clinical trial using the described culture conditions.
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