Stem cell Differentiation media iPSCs or hESCs differentiation into Neuronal cells

Stem cells have the unique ability to self-renew or differentiate themselves into various cell types in response to appropriate signals. These cells are especially important for tissue repair, regeneration, replacement, or in the case of hematopoietic stem cells (HSCs) to differentiate into various myeloid populations. Appropriate signals refer to the growth factor supplements or cytokines that mediate differentiation of various stem cells into the required differentiated form. For instance, HSCs can be differentiated into dendritic cells (with IL-4 and GM-CSF), macrophages (with m-CSF) and MDSCs (with IL-6 and GM-CSF). Human pluripotent stem cells (hPSCs) and induced pluripotent stem cells (iPSCs) can be first cultured in neural differentiation media (GSK3𝛃-i, TGF𝛃-i, AMPK-i, hLIF) to form neural rosettes, which can be differentiated into neural or glial progenitors (finally differentiated into oligodendrocytes). Neural progenitors can be finally differentiated into glutaminergic (dibytyryl cAMP, ascorbic acid) and dopaminergic (SHH, FGF-8, BDNF, GDNF, TGF-𝛃3) neurons. Thus, it is important to first identify the self-renewing cell line: its source and its final differentiation state, followed by the supplements and cytokines required for the differentiation, and final use. Timelines are another thing that is considered. For instance, it takes 7-10 days to form neural rosettes from iPSCs and 3 days to differentiate neural progenitors to neurons. Finally, the stability for stem cell culture media varies. It is advised to make fresh media every time when differentiating HSCs to myeloid populations, whereas neural differentiation media may remain stable for two weeks when stored in dark between 2-8C.

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Found 3 matching solutions for this experiment

Gibco™ DMEM/F-12, GlutaMAX™ supplement

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	DMEM/F12-GLUTAMAX-type medium containing 4% B27 supplement without vitamin A (Invitrogen), 1% N2 supplement (Invitrogen), and 55 M 2-mercaptoethanol (2-ME). To induce forebrain fate, the medium was supplemented with 5µM Y-27632 and 200ng/ml recombinant mouse Noggin (R&D Systems, 1967-NG-025)	After 2 days, EBs were collected and plated onto 10-cm bacterial Petri dishes in 10 ml neuronal medium as above with the addition of B27 supplemented with vitamin A.

Protocol tips
DMEM/F12-GLUTAMAX-type medium containing 4% B27 supplement without vitamin A (Invitrogen), 1% N2 supplement (Invitrogen), and 55 M 2-mercaptoethanol (2-ME). To induce forebrain fate, the medium was supplemented with 5µM Y-27632 and 200ng/ml recombinant mouse Noggin (R&D Systems, 1967-NG-025)
Downstream tips
After 2 days, EBs were collected and plated onto 10-cm bacterial Petri dishes in 10 ml neuronal medium as above with the addition of B27 supplemented with vitamin A.

Manufacturer protocol Publication protocol 1 Relevant paper

Gibco™DMEM/F-12

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	increasing amounts of N2 media (25, 50, 75, 100%) were added to the mTeSR medium every other day whilst maintaining 2.5uM dorsomorphin. N2 medium contains 1X N2 supplement (Invitrogen), 50% DMEM/F12, 50% Neurobasal, 0.075% Bovine serum albumin, and 2mM GlutaMAX (Life Technologies)	Cells were subsequently passaged with trypsin and cultured on matrigel-coated dishes in N2 media with the addition of 20ng/ml bFGF. Rho-associated protein kinase (ROCK) inhibitor Y27632 (10uM, Stemgent) was added to the medium for the first 24 hours. NPs were expanded and maintained in N2 medium with 20ng/ml bFGF. All 2D culture experiments were performed on WIBR3-derived NPs unless otherwise specified.

Protocol tips
increasing amounts of N2 media (25, 50, 75, 100%) were added to the mTeSR medium every other day whilst maintaining 2.5uM dorsomorphin. N2 medium contains 1X N2 supplement (Invitrogen), 50% DMEM/F12, 50% Neurobasal, 0.075% Bovine serum albumin, and 2mM GlutaMAX (Life Technologies)
Downstream tips
Cells were subsequently passaged with trypsin and cultured on matrigel-coated dishes in N2 media with the addition of 20ng/ml bFGF. Rho-associated protein kinase (ROCK) inhibitor Y27632 (10uM, Stemgent) was added to the medium for the first 24 hours. NPs were expanded and maintained in N2 medium with 20ng/ml bFGF. All 2D culture experiments were performed on WIBR3-derived NPs unless otherwise specified.

Manufacturer protocol Publication protocol 1 Relevant paper

Gibco™Neurobasal™ Medium

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
NS cells were grown on poly-L-ornithin and laminin coated plates. Poly-L-ornithine/laminin plates were generated as such: A 20 ug/ml solution of poly-L-ornithine (Sigma) in water was added to plates and plates were incubated at 37°C for 1 hour. The poly-L-ornithine solution was then removed, plates were washed three times with PBS, and then 5 ul/ml solution of laminin (Sigma) in PBS was added to plates and plates were incubated at 37°C for at least 3 hours.	1:1 mix of DMEM/F12:Neurobasal (Invitrogen), 0.5X N2 (Invitrogen), 0.5X B27 (Invitrogen), 1X Glutamax, and 0.1 mM beta-mercaptoethanol, which was supplemented with 20 ng/ul of both EGF and FGF-2 (R&D)

Upstream tips
NS cells were grown on poly-L-ornithin and laminin coated plates. Poly-L-ornithine/laminin plates were generated as such: A 20 ug/ml solution of poly-L-ornithine (Sigma) in water was added to plates and plates were incubated at 37°C for 1 hour. The poly-L-ornithine solution was then removed, plates were washed three times with PBS, and then 5 ul/ml solution of laminin (Sigma) in PBS was added to plates and plates were incubated at 37°C for at least 3 hours.
Protocol tips
1:1 mix of DMEM/F12:Neurobasal (Invitrogen), 0.5X N2 (Invitrogen), 0.5X B27 (Invitrogen), 1X Glutamax, and 0.1 mM beta-mercaptoethanol, which was supplemented with 20 ng/ul of both EGF and FGF-2 (R&D)

Manufacturer protocol Publication protocol 1 Relevant paper

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