Stem cell Differentiation media iPSCs or hESCs differentiation into cerebellar neuroepithelium (NE)

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 2 matching solutions for this experiment

Upstream tips
hESCs were dissociated to single cells and quickly reaggregated using low-cell-adhesion 96-well culture plate with V-bottomed wells (PrimeSurface MS-9096V; Sumitomo Bakelite)
Protocol tips
cerebellar differentiation medium [Iscove’s modified Dulbecco’s medium/Ham’s F-12 1:1, 1 chemically defined lipid concentrate, penicillin/streptomycin, monothioglycerol (450 M) and purified BSA (99% purified by crystallization; Sigma) ](6,000 cells/well) containing growth-factor-free chemically defined medium (gfCDM), insulin (7 ÎŒg/ml), 10 ÎŒM Y-27632, and 10 ÎŒM SB431542 (Tocris Bioscience)
Upstream tips
NMM Contains: DMEM/F12, Glutamax (500 ml, Life Technologies), Neurobasal medium (500 ml, Life Technologies), N2 supplement (1×, Life Technologies), B27 supplement (1×, Life Technologies), insulin (5 Όg ml−1, Imgen), L-glutamine (1.5 mM, Life Technologies), non-essential amino acids (NEAA, 100 ”M, Life Technologies), penicillin/streptomycin (100 U l−1, Life Technologies), beta-mercaptoethanol (10 ”M, Life Technologies). Medium was stored at 4°C and used within 3 weeks.
Protocol tips
Neural Maintenance Medium (NMM)+FGF2 (4 ng ml−1, Peprotech)+RI (10 ”M), and distributed to anti-adhesive (Poly-HEMA, Sigma, P3932-10G) coated 6WPs in a 2 or 3:1 ratio (with 2.5 ml medium per well)
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