Stem cell Differentiation media hESCs or iPSCs differentiation into ovarian follicle/granulosa 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 2 matching solutions for this experiment

Gibco™KnockOut™ DMEM

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
∼5 × 104 undifferentiated hESCs (∼50% confluency in a six-well plate) were seeded onto one well of a six-well plate coated with Matrigel.	differentiation medium (KODMEM supplemented with 10% FBS, 1 mM l-glutamine, 0.1 mM nonessential amino acids, 50 ng ml−1 BMP4 and BMP8a (R&D systems)) for 1 h at 37 °C with 5% CO2.	At day 7, the differentiated hESCs were incubated with differentiation medium containing 0.35 ng ml−1 GDF9:BMP15 heterodimer (prepared as described previously in Matzuk's lab39) and 10 ng ml−1 EGF (R&D) for 1 day, followed by new differentiation medium containing 50 ng ml−1 GDF9 and 25 ng ml−1 BMP15 (R&D systems)

Upstream tips
∼5 × 104 undifferentiated hESCs (∼50% confluency in a six-well plate) were seeded onto one well of a six-well plate coated with Matrigel.
Protocol tips
differentiation medium (KODMEM supplemented with 10% FBS, 1 mM l-glutamine, 0.1 mM nonessential amino acids, 50 ng ml−1 BMP4 and BMP8a (R&D systems)) for 1 h at 37 °C with 5% CO2.
Downstream tips
At day 7, the differentiated hESCs were incubated with differentiation medium containing 0.35 ng ml−1 GDF9:BMP15 heterodimer (prepared as described previously in Matzuk's lab39) and 10 ng ml−1 EGF (R&D) for 1 day, followed by new differentiation medium containing 50 ng ml−1 GDF9 and 25 ng ml−1 BMP15 (R&D systems)

Manufacturer protocol Publication protocol 1 Relevant paper

Gibco™DMEM/F-12

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
Briefly, the iPS cells were stimulated with all-trans-retinoic acid (atRA) for 48 h, then atRA was removed. The iPS cells were cultured in fresh medium supplemented with E2, FSH and human growth hormone (hGH) for 48 h. The iPS cells were then cultured in fresh medium supplemented with anti-Müllerian hormone (AMH), estradiol, FSH and hGH for 48 h. Finally, the iPS cells were cultured in fresh medium supplemented with AMH, estradiol, inhibin α, inhibin β and transforming growth factor-β for 96 h.	Dulbecco's modified Eagle's medium:F12 (1:1) supplemented with 15% KnockOut™ Serum Replacement, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM beta-mercaptoethanol, penicillin (25 U/ml)-streptomycin (925 mg/ml), Reconstituting Human Basic Fibroblast Growth Factor (15 ng/ml), Reconstituting Human Epidermal Growth Factor (15 ng/ml)

Upstream tips
Briefly, the iPS cells were stimulated with all-trans-retinoic acid (atRA) for 48 h, then atRA was removed. The iPS cells were cultured in fresh medium supplemented with E2, FSH and human growth hormone (hGH) for 48 h. The iPS cells were then cultured in fresh medium supplemented with anti-Müllerian hormone (AMH), estradiol, FSH and hGH for 48 h. Finally, the iPS cells were cultured in fresh medium supplemented with AMH, estradiol, inhibin α, inhibin β and transforming growth factor-β for 96 h.
Protocol tips
Dulbecco's modified Eagle's medium:F12 (1:1) supplemented with 15% KnockOut™ Serum Replacement, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM beta-mercaptoethanol, penicillin (25 U/ml)-streptomycin (925 mg/ml), Reconstituting Human Basic Fibroblast Growth Factor (15 ng/ml), Reconstituting Human Epidermal Growth Factor (15 ng/ml)

Manufacturer protocol Publication protocol 1 Relevant paper

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