Biomaterial depots were prepared using well characterized diblock copolypeptide hydrogels that are CNS biocompatible, biodegrade over several weeks in vivo and provide prolonged delivery of bioactive growth factors in CNS tissue for two or more weeks after injection18 (link),19 (link),37 (link),38 (link). Diblock copolypeptide hydrogel K180L20 was fabricated, conjugated with blue fluorescent dye (AMCA-X) and loaded with growth factor and antibody cargoes as described36 (link)–38 (link). Cargo molecules were as follows: Human recombinant FGF2, EGF and GDNF were purchased from PeproTech (Rocky Hill, NJ): (i) Human FGF2 (FGF-basic) (154 a.a.) Cat#100-18B-100UG, Lot#091608 C0617; (ii) Human EGF Cat#AF-100-15-100UG, Lot#0816AFC05 B2317; (iii) Human GDNF Cat#405-10-100UG, Lot#0606B64 A2517. Integrin-function-blocking Hamster anti-rat CD29 monoclonal antibody was purchased from BD Bioscience (San Diego, CA) as a custom order at 10.3mg/ml (product #624084; lot#7165896). Freeze dried K180L20 powder was reconstituted to 3.0% or 3.5% wt/vol in sterile PBS without cargo or with combinations of FGF2 (1.0µg/µl), EGF (1.0µg/µl), GDNF (1.0µg/µl) and anti-CD29 (5µg/µl). Diblock copolypeptide hydrogel formulations were prepared to have G′ (storage modulus at 10 rad s-1) between 75 and 100 Pascal (Pa), somewhat below that of mouse brain at 200 Pa37 (link),38 (link).
Human gdnf
Manufactured by Thermo Fisher Scientific
Sourced in United States
Human GDNF is a recombinant protein that is the human form of Glial Cell Derived Neurotrophic Factor (GDNF). GDNF is a neurotrophic factor that supports the survival and function of various types of neurons.
Automatically generated - may contain errors
Lab products found in correlation
Human egf, by Thermo Fisher Scientific (4 mentions)
Human bdnf, by Thermo Fisher Scientific (3 mentions)
Human bfgf, by Thermo Fisher Scientific (3 mentions)
Human recombinant fgf2, by Thermo Fisher Scientific (2 mentions)
Human fgf2 fgf basic 154 a a, by Thermo Fisher Scientific (2 mentions)
Hamster anti rat cd29 monoclonal antibody, by BD (2 mentions)
Penicillin streptomycin, by Welgene (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
D glucose, by Thermo Fisher Scientific (2 mentions)
Pyruvic acid, by Thermo Fisher Scientific (2 mentions)
Stempro 34 sfm, by Thermo Fisher Scientific (2 mentions)
Progesterone, by Merck Group (2 mentions)
Murine leukemia inhibitory factor, by ProSpec (2 mentions)
Mem vitamins, by Welgene (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
β estradiol, by Merck Group (2 mentions)
Dl lactic acid, by Merck Group (2 mentions)
Heparin, by Thermo Fisher Scientific (1 mentions)
9 protocols using human gdnf
1
Biodegradable Hydrogel Biomaterial Depots
2
Fibrin-based Nerve Guidance Conduits with GDNF
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Fibrinogen solutions (8 mg/ml) were prepared by dissolving human plasminogen-free fibrinogen (Calbiotech Inc., Spring Valley, CA) in deionized water for 1 h at 37°C, then dialyzing the solution against 4 L of Tris-buffered saline (TBS) containing 33 mM Tris, 8 g/L NaCl, and 0.2 g/L KCl (Sigma-Aldrich, St. Louis, MO)1 . The fibrinogen solution was sterile filtered using 5.0 and 0.22 μm syringe filters; spectrophotometry confirmed the concentration of fibrinogen. Just prior to surgery, fibrinogen, TBS, 50 mM CaCl2 in TBS, 20 U/ml thrombin, 25 mg/ml a2PI1–7-ATIII124–134 peptide, 45 mg/ml heparin, and 100 ng/ml human GDNF (Peprotech, Rocky Hill, NJ) were mixed in an Eppendorf tube (Lee et al., 2003 (link)). Immediately after mixing, 30 μl of the fibrin-based delivery system containing GDNF was injected into the nerve guidance conduits of Groups III and V. Prior to surgical implantation, applied fibrin matrices polymerized for 5 min.
3
Differentiation of Cortical Neurons from hESCs
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Cortical neurons were differentiated from human embryonic stem cells (hESCs; H9) according to the protocol by Shi et al. (2012) (link). Pluripotent stem cell colonies at 80% confluence were used as the starting material. After a 10-day neural induction with 10 μM SB431542 (Tocris Bioscience no. 1614) and 1 μM Dorsomorphin (Sigma no. P5499), neuroepithelial cells were passaged and cultured in neural maintenance medium, according to the protocol, on ECM (1:100, Sigma no. E1270) coated plates for 14–18 additional days. After a first passage by cutting and replating the neural rosettes, neural stem cells (NSCs) were expanded in maintenance medium and passaged with Accutase (StemPro, Life Technologies no. A1110501). To promote terminal differentiation, NSCs were plated on poly-L -ornithine/laminin-coated plates (0.1 mg/ml poly-L -ornithine hydrobromide, 30,000–70,000 kDa, Sigma no. P3655, and 5 μg/ml mouse laminin, Invitrogen no. 23017-015) and cultured in neural maintenance medium in the presence of 20 ng/ml human BDNF (Peprotech no. 450-02), 20 ng/ml human GDNF (Peprotech no. 450-10), 200 nM ascorbic acid (Sigma no. A4544), 0.5 mM dibutyryl cAMP sodium salt (Sigma no. D0627) and 1 μg/ml laminin.
4
Biodegradable Hydrogel Biomaterial Depots
5
Lentivirus-mediated GFP Transduction and Maturation of Neural Stem Cells
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NESC plated in T75 flasks covered with Matrigel were infected with lentivirus encoding for GFP as previously reported5 . Briefly, 3 × 106 cells were infected with lentivirus (1.33 × 103 ng of p24 antigen) and 24 h later half of the culture medium was replaced to dilute the virus. On the second day, the lentiviral vectors were completely removed by changing the medium. Five days before transplantation, cells were submitted to a maturation protocol with the N2B27 medium supplemented with 10 ng/ml human BDNF (Peprotech), 10 ng/ml human GDNF (Peprotech), 1 ng/ml human TGFβ-3 (Peprotech), 200 μM Ascorbic Acid, and 250 μM dbcAMP. For transplantation, cells were prepared in Hank's Balanced Salt Solution HBSS (Sigma) at 150 000 cells/2 μl.
6
Differentiation of PSCs to Striatal Neurons
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Cells were differentiated to a striatal neuron fate using a published method [44 (link)]. Briefly, PSC monolayers were first directed to a forebrain neural progenitor fate by way of the presence of N2 and B27 supplements (Gibco, Thermo Fisher Scientific) and BMP and SMAD inhibitors (100 nM LDN193189 (Sigma-Aldrich, Merck); 200 nM dorsomorphin (Cambridge Biosciences); 10 μM SB431542 (Cambridge Biosciences)) over a 9-10 day period. Thereafter, patterning to a lateral ganglionic eminence/striatal fate was achieved by the addition of 25 ng/ml activin A (PeproTech), which was maintained throughout the remainder of the differentiation process. The addition of 10 ng/ml human BDNF (PeproTech), 10 ng/ml human GDNF (PeproTech) and vitamin A-containing B27 for 10 days from day 26 post-induction onwards aided maturation of these striatal precursors to a more mature striatal neuron identity, resulting in cultures expressing markers of striatal GABAergic neurons, including a proportion expressing a marker of full maturity, DARPP32.
7
Establishment of Transgenic SSCs from Mice
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Establishment of SSCs from Oct4-GFP/LacZ and Oct4-GFP transgenic mice (C57BL/6 background) was described previously (Ko et al. 2009 (link); Kanatsu-Shinohara et al. 2011 (link); Han et al. 2012 (link)). SSC medium for expansion was composed of StemPro-34 SFM (Gibco, Carlsbad, CA, USA) with the following supplements: StemPro (Gibco), 1× N2 (Gibco), 6 mg/ml d-(+)-glucose (Gibco), 30 mg/ml pyruvic acid (Gibco), 1 μl/ml DL-lactic acid (Sigma-Aldrich, Saint Louis, MO, USA), 5 mg/ml bovine serum albumin (BSA; Gibco), 1% fetal bovine serum (FBS; Gibco), 2 mM L-glutamine (Gibco), 50 μM β-mercaptoethanol (Gibco), 1×penicillin/streptomycin (Welgene, Gyeongsan, Korea), 1× minimal essential medium (MEM) with non-essential amino acids (Gibco), 1× MEM vitamins (Welgene), 30 ng/ml β-estradiol (Sigma), 60 ng/ml progesterone (Sigma), 20 ng/ml human EGF (Peprotech, Rocky Hill, NJ, USA), 20 ng/ml human bFGF (Peprotech), 20 ng/ml human GDNF (Peprotech), and 103 U/ml murine leukemia inhibitory factor (Prospec, Rehovot, Israel).
8
Establishment of SSCs from Oct4-GFP/LacZ Mice
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Establishment of SSCs from Oct4-GFP/LacZ transgenic mice (C57BL/6 background) was described previously (Ko et al., 2009 (link); 2010 (link); 2012 (link)). SSC medium for expansion was composed of StemPro-34 SFM (Gibco) with the following supplements: StemPro supplement (Gibco), 1× N2 supplement (Gibco), 6 mg/ml d-(+)-glucose (Gibco), 30 mg/ml pyruvic acid (Gibco), 1 μl/ml DL-lactic acid (Sigma-Aldrich), 5 mg/ml bovine serum albumin (BSA; Gibco), 1% fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 50 μM β-mercaptoethanol (Gibco), 1×penicillin/streptomycin (Welgene), 1× minimal essential medium (MEM) non-essential amino acids (Gibco), 1× MEM vitamins (Welgene), 30 ng/ml β-estradiol (Sigma-Aldrich), 60 ng/ml progesterone (Sigma-Aldrich), 20 ng/ml human EGF (Peprotech), 20 ng/ml human bFGF (Peprotech), 20 ng/ml human GDNF (Peprotech), and 103 U/ml murine leukemia inhibitory factor (Prospec).
9
Differentiation of hiPSCs and hESCs into SSCLCs
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Human iPSC lines have been described previously [29, 30] , including a normal cell line and two cell lines from unrelated NOA patients with unknown causes and testicular histology. hiPSC lines and H1 ESCs were maintained in mTeSR1 medium (STEMCELL Technologies) at 37℃ and 5% CO 2 . The medium was changed every day. Cells were passaged every 3 to 4 days using 0.5 mM EDTA. An amount of 10 μM ROCK inhibitor Y-27632 (Selleck) was added into the medium for 24 h after every passage.
For hiPSCs and hESCs differentiation into SSCLCs, cells were rstly seeded on Matrigel (Corning)-coated 12 or 24-well plates in mTeSR1 medium containing 10 μM ROCK inhibitor, and the medium was replaced by SSCLC induction medium on the second day when cells reached 80%-90% con uence. The components of SSCLC induction medium were based on previous studies with some modi cations [11, 16] , which contained a-MEM, 3% Knockout serum replacement, 1% GlutaMax, 1% 100 × Insulin-Transferrin-Selenium-X, 0.2% chemically de ned lipid concentrate (all from Thermo Fisher Scienti c), 20 ng/mL human GDNF, 1 ng/mL human b-FGF (all from Peprotech), 100-200 μg/mL VPA and/or 100-200 μg/mL VC (all from Sigma Aldrich). The concentrations of human GDNF, human b-FGF, VPA and VC varied with the purposes of experiments. The SSCLC induction medium was changed every day until the cells were ready for analysis.
For hiPSCs and hESCs differentiation into SSCLCs, cells were rstly seeded on Matrigel (Corning)-coated 12 or 24-well plates in mTeSR1 medium containing 10 μM ROCK inhibitor, and the medium was replaced by SSCLC induction medium on the second day when cells reached 80%-90% con uence. The components of SSCLC induction medium were based on previous studies with some modi cations [11, 16] , which contained a-MEM, 3% Knockout serum replacement, 1% GlutaMax, 1% 100 × Insulin-Transferrin-Selenium-X, 0.2% chemically de ned lipid concentrate (all from Thermo Fisher Scienti c), 20 ng/mL human GDNF, 1 ng/mL human b-FGF (all from Peprotech), 100-200 μg/mL VPA and/or 100-200 μg/mL VC (all from Sigma Aldrich). The concentrations of human GDNF, human b-FGF, VPA and VC varied with the purposes of experiments. The SSCLC induction medium was changed every day until the cells were ready for analysis.
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