Bone marrow stem cells (Cyagen Biosciences Inc., USA), NG-108 cells (ATCC, USA), fetal bovine serum (Gibco, USA), Dulbecco’s Modified Eagle Medium (DMEM)/F12 Culture medium (Gibco), trypsin (Sigma, USA), penicillin (Gibco), streptomycin (Gibco), beta-mercaptoethanol (Amresco, USA), all trans-retinoic acid (Sigma), forskolin (Peprotech, UK), basic fibroblast growth factor (bFGF) (Peprotech), platelet-derived growth factor (PDGF) (Peprotech), recombinant human heregulin-b1 (heregulin-b1) (Peprotech), S-100 antibodies (Abcam, USA), glial fibrillary acidic protein (GFAP) antibodies (Abcam), β-actin antibody (Proteintech Group, USA), RIPA cell lysate (Biotime Company, CHN), BCA protein determination reagent (Biotime Company), SDS PAGE gel preparation kit (Biotime Company), DAB light liquid (Biotime Company), goat anti-rabbit IgG2 (Biotime Company), and Tris-buffered saline plus Tween 20 (TBST) (Biotime Company) were all used for the current study. Annexin V-FITC/PI apoptosis and cell cycle kit(Liankebio company,CHN),Mitochondria Staining Kit(JC-1) (Liankebio company).
Platelet derived growth factor
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Platelet-derived growth factor is a protein involved in cell growth and division. It plays a role in the regulation of cell proliferation, migration, and differentiation. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (8 mentions)
Forskolin, by Merck Group (5 mentions)
β mercaptoethanol, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Forskolin, by Thermo Fisher Scientific (2 mentions)
Neurotrophin 3, by Thermo Fisher Scientific (2 mentions)
Trace element b, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Ng 108 cells, by American Type Culture Collection (1 mentions)
β mercaptoethanol, by Avantor (1 mentions)
S 100 antibodies, by Abcam (1 mentions)
Trypsin, by Merck Group (1 mentions)
β actin antibody, by Proteintech (1 mentions)
Mercaptoethanol, by Merck Group (1 mentions)
Adipogenic differentiation medium, by Thermo Fisher Scientific (1 mentions)
Osteogenic differentiation medium, by Thermo Fisher Scientific (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Neural survival factor 1, by Lonza (1 mentions)
14 protocols using platelet derived growth factor
1
Differentiation and Characterization of Bone Marrow Stem Cells
2
Multilineage Differentiation of DPSCs
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Conventional condition-cultured DPSCs were induced into osteogenic, glial and adipogenic lineages. The cells (5,000 cells/well) were cultured on plain 8-well chambers [except for glial differentiation induction where the wells were coated with 4 µg/ml of laminin (Gibco, CA, USA)] for 1 week before differentiation induction. Osteogenic differentiation was induced by incubating the cells with osteogenic differentiation medium (Gibco, NY, USA) for 3 weeks. Thereafter, immunofluorescence of osteocalcin was performed. For glial differentiation, the cells were supplemented with αMEM containing 1 mM mercaptoethanol (Sigma, Germany) without FCS for 24 h and then for 3 days with 20% FCS αMEM containing 35 ng/ml of retinoic acid (Sigma, China). Thereafter, the cells were incubated in 20% FCS-αMEM supplemented with 5 µM forskolin (Sigma, USA), 10 ng/ml of bFGF, 5 ng/ml of platelet-derived growth factor (PDGF) (Peprotech, London, UK), and 200 ng/ml of recombinant human neuregulin-ß1 (Sigma, USA) for the following 3 weeks. Immunofluorescence detection of the Schwann cell marker S100ß was then performed. Finally, the induction of adipogenic lineage differentiation was achieved by culturing cells with adipogenic differentiation medium (Gibco, NY, USA) for three weeks before performing lipidtox staining.
3
Isolation and Characterization of NPCs
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Isolated NPCs were seeded on poly-d -lysine coated flasks with neuronal media containing Neurobasal media, bovine serum albumin (BSA) (Sigma–Aldrich, USA), N2 supplement (Invitrogen, San Diego, CA, USA), and Neural Survival Factor-1 (Lonza, Charles City, IA), supplemented with 10 ng/ml Platelet-derived growth factor (PDGF) (Peprotech, Rocky Hill, NJ, USA) and 10 ng/ml Brain-derived growth factor (BDNF) (Peprotech, Rocky Hill, NJ, USA). Half media was changed till maturation on every alternate day, followed by characterization for neuronal markers, and 99% of the cells were Tuj-1 and Map2 positive.
4
Multilineage Differentiation of iNSCs
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For neuronal differentiation of iNSCs and NSCs, 5×104 cells were plated onto polyornithine- and laminin-coated 24-well plates (Sigma–Aldrich), cultured in DMEM/F12 (1:1), B27, EGF, and bFGF for 2 days, and differentiated in B27 and DMEM/F12 (1:1) for 12 days. For electrophysiological analysis, cells were cultured for an additional 18 days in N2, B27 medium supplemented with 1% serum. For astrocytic differentiation, 5×104 cells were split onto polyornithine-coated 24-well plates and cultured in B27, DMEM/F12 medium plus 5% FBS without FGF and EGF for 5–8 days. For oligodendrocyte differentiation, 5×104 cells were plated onto polyornithine- and laminin-coated 24-well plates and cultured in 10 ng/mL bFGF, 10 ng/mL platelet-derived growth factor (PDGF; Peprotech, Beijing, China), and 10 nM forskolin (Sigma–Aldrich) for 5 days, and then in 200 nM ascorbic acid (Sigma–Aldrich) and 30 ng/mL T3 (Sigma–Aldrich) for 5 days.
5
Isolation and Culture of Oligodendrocyte Progenitor Cells
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Oligodendrocyte progenitor cells (OPC) were isolated from cortical astrocyte monolayers generated from postnatal day 1 (P1) SD pups by differential adhesion as previously described (Miron et al., 2013 ; Noble & Murray, 1984 ) . OPCs were maintained in serum‐free DMEM‐BS (adapted from Bottenstein & Sato, 1979 ) containing 0.5 mg/ml insulin in 10 mM HCL (Sigma, UK), glutamine (100 mM, Sigma), human transferrin (0.1 mg/ml, Sigma) and gentamycin (100 mg/ml, Sigma), supplemented with the growth factors; fibroblast growth factor (FGF‐2) at 10 ng/ml and platelet derived growth factor (PDGF) at 2 ng/ml (both Peprotech, UK). The isolated OPCs were plated on poly‐l ‐lysine (PLL, 13 μg/ml, Sigma) coated glass coverslips (VWR) in a 24‐well plate at a density of 5,000 cells in 50 μl drop and allowed to attach. They were maintained in DMEM‐BS containing PDGFα and FGF2 for 5 days and then switched to DMEM‐BS lacking growth factors and with or without mHeps at a concentration of 1 ng/ml. Cultures were used for proliferation, morphology, and differentiation assays.
6
Differentiation of Adipose-Derived Stem Cells into Schwann Cells
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ASCs were harvested from Sprague–Dawley rats (Janvier, France) as described earlier(de Luca et al., 2018 ) and maintained in Modified Eagle Medium (α‐MEM; Invitrogen, UK) containing 1% (v/v) streptomycin/penicillin solution and 10% (v/v) FBS. ASCs were differentiated at early passages, i.e., 4–6. When the cells attained the subconfluent sate, growth medium was replaced with fresh medium supplemented with 1 mM β‐mercaptoethanol (Sigma‐Aldrich, UK) for 24 h. Cells were then washed and fresh medium supplemented with 35 ng/mL all‐transretinoic acid was added. 72 h later, cells were washed and differentiation medium, enriched with 5 ng/ml platelet‐derived growth factor (PDGF; PeproTech, UK), 10 ng/mL basic fibroblast growth factor (bFGF; PeproTech, UK), 40 ng/mL of recombinant neuregulin‐β1 (NRG1‐β1), (R&D Systems, UK) and 14 μM forskolin, was added. Cells were incubated for 14 days under these conditions and differentiation growth medium was exchanged at an interval of 72 h. To confirm the effectiveness of the differentiation of stem cells, resulting SCs phenotype was confirmed by specific markers S100 and GFAP (Caddick et al., 2006 ; Kingham et al., 2007 ).
7
Differentiation of Bone Marrow Mesenchymal Stem Cells
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After sub-culturing at the concentration of 106 cells/cm2, BMMSCs and MT-BMMSCs were incubated in a serum-free DMEM containing 1 mM beta-mercaptoethanol (Sigma, USA) for 24 h. Then the culture media were replaced by DMEM containing 10% FBS and 35 ng/ml all-trans-retinoic acid (Sigma, USA) for three days. Finally, for further 10 days, the cells were placed in an inducer medium containing DMEM, 10% FBS and trophic factors, including 5 ng/ml platelet-derived growth factor (Peprotech, UK), 10 ng/ml beta fibroblast growth factor (Peprotech, UK), 5 µM forskolin (Calbiochem, Canada) and 200 ng/ml hergulin (R&D Systems, USA).
8
Schwann-like Cell Differentiation from Rat AMSCs
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AMSCs were differentiated into SLCs using an established protocol by Kingham16 (link),17 (link). The Schwann-like cells were derived from white male rat AMSCs. In the third passage, the AMSCs cells were differentiated into a Schwann-like cell-differentiated AMSCs (SLCs/dAMSCs) phenotype in the first two steps. First, the growth medium was replaced with a medium complemented with 1 mM-mercaptoethanol (Scharlau Chemicals) for 24 h, followed by administration of 35 ng/ml all-trans-retinoic acid (Sigma-Aldrich) for 72 h. After that, the cells were managed with a differentiation medium containing a growth medium complemented with 5 ng/ml platelet-derived growth factor (PeproTech), 10 ng/ml basic fibroblast growth factor (bFGF) (PeproTech), forskolin 14 M (Sigma -Aldrich) ) and 252 ng/ml neuregulin-1 (R&D System) for at least 14 days prior to characterization. The selection of added growth factors is based on their role in modulating the development and survival of Schwann cells18 (link). The Schwann-like cell-differentiated AMSCs was confirmed by Glial Fibrilar Acid Protein (GFAP) and S100 protein expression immunocytochemical examination, and used in this study were cells at passages 3–6.
9
Differentiation of Adipose-Derived Stem Cells
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As previously described (Kingham et al., 2007 (link)), passage 2 AD-MSC were plated at 20 to 30% confluency and treated with 1 mM b-mercaptoethanol (Sigma-Aldrich) for 24 h, prior to 72 h of exposure to 35 ng/mL all-trans-retinoic acid (Sigma-Aldrich). The differentiation media (d-aMEM) was then added ([aMEM) + 14 μM forskolin (Sigma-Aldrich) + 5 ng/mL platelet-derived growth factor (Peprotech EC, London, United Kingdom) + 192 ng/mL glial growth factor-2 (GGF-2) (Acorda Therapeutics, Ardsley, NY, United States) + 10 ng/mL basic fibroblast growth factor (Peprotech EC)]. Cells were split at the fourth and tenth days of differentiation, and cells were considered differentiated (dASCs) after 14 days, as previously reported (Kingham et al., 2007 (link)). Cells at passage 4 to 6 were used in co-culture experiment involving dASCs.
10
Clonal Mesensphere Formation Protocol
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For clonal mesensphere formation, BMSCs were plated at clonal density (∼1,000 cells/cm2) in ultralow adherent 24-well plates (Corning) as previously described (Mendez-Ferrer et al., 2010 (link), Pinho et al., 2013 (link)). The growth medium contained 15% chicken embryo extract (Fisher Scientific), 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, 1% N2 and 2% B27 supplements (Gibco), basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, platelet-derived growth factor, and oncostatin M (Peprotech) (20 ng/ml) in DMEM/F12 (1:1)/human endothelial (Gibco) (1:2). The cultures were maintained at 37°C in a 5% CO2 water-jacketed incubator and left untouched for 1 week to prevent cell aggregation in low-density cultures. Half of the medium was changed weekly. The mesensphere colonies were detached with trypsin, and the cells were mechanically dispersed and re-plated back into ultralow adherent plates with culture medium. Secondary mesenspheres were counted after 7 days in culture.
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