iPSCs were manually dissected from MEF, then grown in suspension culture for 4 days in EB medium supplemented with 2 μM dorsomorphin (R&D Systems, 3096; Minneapolis, MN, USA) and 5 μM SB431542 (R&D Systems, 1614). Medium was then changed to neural induction medium consisting of DMEM/F12, 1 × N2 supplement (Invitrogen, 17502-048), 1 × NEAA, 2 mM glutamax, 0.1 mM β-mercaptoethanol, 2 μM dorsomorphin, 5 μM SB431542 and 20 ng ml−1 FGF2 for an additional 3 days in suspension. Neurospheres were then planted on laminin (Sigma-Aldrich, L2020)-coated culture dishes for rosette formation. Rosettes were manually picked 2–3 times, dissociated in Accutase then re-plated on poly-D-ornithine-laminin-coated substrates for neuronal differentiation in medium composed of neural basal medium (Invitrogen, 21103–049), 1 × B27 supplement (Invitrogen, 17504–044), 1 mM glutamine, 1% NEAA and 1 × Penicillin/Streptomycin for an additional 4, 8 or 12 weeks. Medium was changed every other day. To determine whether dorsal–ventral fate could be respecified, cells were grown with the Smoothened agonist (Hedgehog pathway activator) purmorphamine (1 μm; Cayman Chemical, Ann Arbor, MI, USA, 10009634), or with the dorsalizing agent lithium chloride (LiCl; 1 mM ; Sigma-Aldrich). Controls were exposed to DMSO (carrier) alone.42 (link),43 (link)
Dorsomorphin
Manufactured by R&D Systems
Sourced in United States
Dorsomorphin is a small molecule that inhibits the BMP (Bone Morphogenetic Protein) signaling pathway. It acts as a selective inhibitor of the BMP type I receptors ALK2, ALK3, and ALK6.
Automatically generated - may contain errors
Lab products found in correlation
Sb431542, by R&D Systems (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Purmorphamine, by Cayman Chemical (1 mentions)
Sb431542, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Mlpoly l ornithine, by Merck Group (1 mentions)
Rock inhibitor y 27632, by Bio-Techne (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Accutase, by Corning (1 mentions)
Laminin, by Corning (1 mentions)
Laminin coated, by Merck Group (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
N2 neuroplex, by Gemini Bio (1 mentions)
Gem21 neuroplex, by Gemini Bio (1 mentions)
Heparin sodium, by Merck Group (1 mentions)
Chir99021, by Selleck Chemicals (1 mentions)
7 protocols using dorsomorphin
1
Neuronal Differentiation from iPSCs
2
Differentiation of PSCs to NPCs
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Differentiation was performed as previously described (Chailangkarn et al, 2016 ). After seven days, mTeSR1 used to feed PSCs was replaced with N2 medium [DMEM/F12 (Life Technologies, Carlsbad, CA, USA), 0.5× N2 (Life Technologies), 1% penicillin/streptomycin (P/S; Life Technologies), 1 μM Dorsomorphin (Dorso; R&D Systems, Minneapolis, MN, USA), and 10 µM SB431542 (SB; Stemgent, Cambridge, MA, USA)] for 1–2 days, after which embryoid bodies (EBs) were formed by scraping PSC colonies and culturing them in shaker suspension (95 rpm at 37°C) for eight days. EBs were dissociated and plated on a Matrigel‐coated dish in N2B27 medium [N2 medium with 0.5× B27‐Supplement (Life Technologies) and 20 ng/ml FGF‐2]. Emerging rosettes were picked manually, dissociated in Accutase (Life Technologies), and seeded on poly‐L‐ornithine/laminin plates. Emergent NPCs were expanded and maintained in N2B27 medium with feeding on alternate days; all NPCs used for neurons were passage 5–20. FGF‐2 was withdrawn from the medium to induce neuronal differentiation, considered Day 0 of differentiation.
3
Neural Induction from iPSCs
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iPSCs were cultured
on Matrigel in TesR-E8 for 4 days. Cell clusters were lifted from
the plates using L7 Passaging Solution (Lonza), and suspended in EB
medium (DMEM/F12, 20% KnockOut Serum Replacement (Gibco), 1% NEAA,
2 mM GlutaMAX, 0.1 mM β-mercaptoethanol, 2 μM dorsomorphin
(R&D Systems) and 5 μM SB431542 (R&D Systems)) for 4
days, with 10 μM ROCK inhibitor Y-27632 (Tocris) for the first
day. Then medium was replaced with neural induction medium consisting
of DMEM/F12, 1X N2 supplement (Gibco), 1% NEAA, and 2 mM GlutaMAX
for another 3 days. The neurospheres were plated onto 100 μg/mL
poly-l -ornithine (Sigma)- and 10 ng/mL laminin (Corning)-coated
culture dishes. Rosettes were grown and picked after 7 days, dissociated
with Accutase (Corning) into single NPCs, and reseeded onto Matrigel-coated
plates in NPC maintaining medium (Neurobasal Medium (Gibco), 1×
B27 minus vitamin A supplement (Gibco), 2 mM GlutaMAX, 1% NEAA, 1×
penicillin/streptomycin and 20 ng/mL FGF2).
on Matrigel in TesR-E8 for 4 days. Cell clusters were lifted from
the plates using L7 Passaging Solution (Lonza), and suspended in EB
medium (DMEM/F12, 20% KnockOut Serum Replacement (Gibco), 1% NEAA,
2 mM GlutaMAX, 0.1 mM β-mercaptoethanol, 2 μM dorsomorphin
(R&D Systems) and 5 μM SB431542 (R&D Systems)) for 4
days, with 10 μM ROCK inhibitor Y-27632 (Tocris) for the first
day. Then medium was replaced with neural induction medium consisting
of DMEM/F12, 1X N2 supplement (Gibco), 1% NEAA, and 2 mM GlutaMAX
for another 3 days. The neurospheres were plated onto 100 μg/mL
poly-
culture dishes. Rosettes were grown and picked after 7 days, dissociated
with Accutase (Corning) into single NPCs, and reseeded onto Matrigel-coated
plates in NPC maintaining medium (Neurobasal Medium (Gibco), 1×
B27 minus vitamin A supplement (Gibco), 2 mM GlutaMAX, 1% NEAA, 1×
penicillin/streptomycin and 20 ng/mL FGF2).
4
Generation of Functional Neurons from iPSCs
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IPSC medium was changed to N2 medium comprising DMEM/F12 with l -Glutamine and 15 mm HEPES, 1 × N2 NeuroPlex (Gemini Bio-Products, West Sacramento, CA, USA), 1 μm dorsomorphin (R&D System, Minneapolis, MN, USA) and 10 μm SB431542 (Stemgent, Lexington, MA, USA) for 2 days. Next, cells were grown in suspension for 7 days in N2 medium. The formed EBs were gently dissociated and plated onto Matrigel-coated dishes using neural induction (NI) medium (DMEM/F12 with l -Glutamine and 15 mm HEPES, 0.5 × N2 NeuroPlex, 1 × Gem21 NeuroPlex (Gemini Bio-Products) and 20 ng ml−1 bFGF)). Rosettes that emerged were manually picked, dissociated with Accutase (Life Technologies) and re-plated onto 10 μg ml−1 Poly-l -ornithine and 5 μg ml−1 laminin-coated (from Sigma-Aldrich and Life Technologies, respectively) plates. Homogeneous populations of neural progenitor cells (NPC) were expanded using NI medium. The differentiation into neurons was performed upon bFGF withdrawal and addition of 5 μM ROCK inhibitor (Y-27632, Calbiochem, La Jolla, CA, USA). Cells were cultivated for 4 weeks with media changes every 3–4 days.
5
hESC-Derived Mesenchymal Stem Cell Differentiation
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MSC differentiation was performed as previously described 19 (link), 20 (link). In brief, hESCs were dissociated with TrypLE (Thermo, 12605010) and plated in the density of 90,000 cells/cm2 in E8 medium supplemented with ROCK inhibitor Y27632 (Stemgent, 04-0012, 10 μM) for 24 h. Cells were treated with Essential 6 (E6) medium (Thermo, A1516401) supplemented with sodium heparin (Sigma, H3149, 22.5 ng/ml), bFGF (Thermo, PHG0261, 10 ng/ml), CHIR99021 (Selleck, S1263, 1 μM), SB431542 (Stemgent, 04-0010, 10 μM) and dorsomorphin (R&D, 3093, 1 μM). Cells were split at a 1:6 ratio when they became confluent. Fifteen days later, cells were cultured in MSC medium for continued differentiation to MSCs for 5 more days. Following routine characterization for MSC markers and tri-lineage differentiation 66 (link), the resulting cells were designated as EMSCs at passage 0 (p0). EMSCs within 10 passages were used in this study.
6
Embryonic Tongue Mesenchyme Isolation
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The E11.5 tongue swellings were dissected from the mandible and the mesenchyme was separated from the epithelium, as previously described (Liu et al., 2008 (link)). At this stage, the mesenchyme is primarily populated by NC-derived cells (Han et al., 2012 (link)) and very few non-NC-derived myoprogenitors are present in the tongue mesenchyme (Han et al., 2012 (link)). Separated mesenchyme was then transferred to a culture dish, cut into small pieces and cultured in a humidified CO2 incubator at 37°C in a serum-free medium, i.e. a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient F12 (11320033, DMEM/F12, Gibco) containing 50 μg/ml gentamicin sulfate (15750060, Gibco). After 1 day in culture, the medium was replaced with fresh medium and continued to culture for 2 days. Culture medium was collected as conditioned medium. To inhibit the p-Smad1/5/8 activity, dorsomorphin (3093, R&D Systems) was added at 30 µg/ml to the cultures of mesenchyme cells from wild-type mouse tongue.
7
Cortical Organoid Generation from PSCs
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