View source for Embryoid body to melanocyte

{{TimeCourse
|Time_Course=
|collaborators=Meenhard Herlyn
|description=human_H9
|libraryids=CNhs12822,CNhs12823,CNhs12895,CNhs12896,CNhs12897,CNhs12948,CNhs12898,CNhs12899,CNhs12900,CNhs12901,CNhs12902,CNhs12903,CNhs12904,CNhs12905,CNhs12825,CNhs12826,CNhs12827,CNhs12828,CNhs12829,CNhs12830,CNhs12831,CNhs12832,CNhs12833,CNhs12834,CNhs12835,CNhs12836,CNhs12906,CNhs12907,CNhs12908,CNhs12909,CNhs12910,CNhs12911,CNhs12951,CNhs12949,CNhs12912,CNhs12914,CNhs12915,CNhs12916,CNhs12917,CNhs12918,CNhs12919,CNhs12950
|page_name=human_H9
|series=IN_VITRO DIFFERENTIATION SERIES
|species=Human (Homo sapiens)
|zenbu_config=http://fantom.gsc.riken.jp/zenbu/gLyphs/#config=NZn_vDZIH9Kum5ikjdUorB
|TCOverview=Melanocytes are pigment producing cells that reside in and supply melanin to the skin and eyes. These specialized cells are developmental derived from the neural crest and undergo programmed differentiation throughout migration and terminal differentiation upon reaching their final residential destination. While most melanocyte development studies have been assessed in avian and murine models, capturing their differentiation regulatory networks has been difficult due their vast and speedy migration throughout the embryo. Very few studies have documented human melanocyte development. In an effort to reconcile these discrepancies we have developed a method to efficiently differentiate human embryonic stem cells to terminally differentiated pigmented melanocytes (1, 2) Utilizing this differentiation approach we hope to gain insight into pathways active during human melanocyte differentiation.<br>
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References:<br>
1. Defining the conditions for the generation of melanocytes from human embryonic stem cells.<br>
[1] Fang D, Leishear K, Nguyen TK, Finko R, Cai K, Fukunaga M, Li L, Brafford PA, Kulp AN, Xu X, Smalley KS, Herlyn M. Stem Cells. 2006 Jul;24(7):1668-77.<br>
2. Embryonic stem cells as a model for studying melanocyte development.<br>
[2] Zabierowski SE, Herlyn M. Methods Mol Biol. 2010;584:301-16.<br>
|TCSample_description=For the FANTOM5 study the human embryonic stem cell line H9 [WA09 (MEF platform), WiCell Research Institute] was used to study human melanocyte development. In this model, human embryoid bodies (H9EB) were first generated from undifferentiated H9ES for 4 days in suspension culture conditions. H9EBs were then harvested and differentiated on fibronectin-coated flasks in melanocyte differentiation media (Mel-1). The exact protocol has been published by Zabierowski and Herlyn (2). Briefly 70 – 80% confluent feeder-based ES colonies grown in 6-well plates were gently scraped with a cell lifter into T25 flasks (one 6 well plate per T25) and incubated overnight with EB medium at 37oC. The next day cells were transferred into new T25 flasks with fresh EB medium taking care not to disrupt cell clusters. 2/3 of the EB medium was replenished every day for the next 3 days. On day 4 (day 0 of time course) embryoid bodies were harvested and carefully transferred onto 10 ng/ml fibronectin coated T25 flasks with Mel-1 differentiation medium. 2/3 of the differentiation medium was replenished every other day. On day 24 cells were dissociated into single-cells with trypsin/EDTA treatment and replated onto fresh fibronectin-coated T25 flasks (1:1 passage) in Mel-1 without TPA. 2/3 Mel-1 medium without TPA was replenished every 2-3 days. RNA samples were harvested from T25 flasks by direct lysis on days 1, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 and 42 following the QIAGEN RNeasy Kit protocol. RNA yielded from all samples was stored at -80 C. This time course was repeated in triplicate. For each replicate, H9EB (day 0, embryoid bodies prior to plating in melanocyte differentiation media) and undifferentiated H9ES cells were included as negative controls. Additionally, 3 different human foreskin derived melanocyte cell lines were included as positive controls.
Media: a)	Embryoid body (EB) medium (100 ml): 80 ml DMEM/F-12 (Invitrogen #11330-032), 20 ml Knockout-Serum Replacer (Invitrogen #10828-028), 1 ml 200 mM L-glutamine (Invitrogen #21051-024), + 0.7 ml b-mercaptoethanol/1 ml L-glutamine, 1 ml nonessential amino acids 100X (Invitrogen #11140).
b)	Mel-1 medium for melanocyte differentiation (100 ml): 20 ml Dexamethasone (Sigma #D-2915 – make up to 0.25 M in water and store at –20 C), 1 ml ITS Liquid Medium Supplement (Sigma #I-3146), 1 ml Linoleic Acid-BSA (Sigma #L-9530), 30 ml DMEM-Low Glucose (Invitrogen #11885),20 ml MCDB201, 1 ml L-ascorbic acid (Sigma #A-4403), 50 ml Wnt3a conditioned medium), 1 ml Stem Cell Factor (Fitzgerald Industries #RDI-118B-218 – make up to 10 mg/ml in 0.1% BSA in 1X DPBS and store stock at –70 C), 100 ml Basic Fibroblast Growth Factor (Fitzgerald Industries #RDI-118B-218 – make up to 4 mg/ ml in 0.1% BSA in 1X DPBS and store stock at –70oC), 100 ml Endothelin-3 (American Peptide Co. #88-5-10 – make up264 mg/ml in 0.1% BSA in 1X DPBS and store stock at –70 C), 150 ml Cholera toxin (Sigma #C-3012 – make up to 3.32 mg/ml in 0.1% BSA in 1X DPBS and store stock at 4 C), 12.5 ml TPA (Sigma #P-1583 – make up to 250 mg/ml in 0.1% BSA in 1X DPBS and store stock at –20 C).
c)	L-Wnt3A conditioning medium: 1% FBS (1000 ml): 900 ml DMEM (Cellgro #10-017-CM), 10 ml FBS.
d)	L-Wnt3A conditioned medium: Confluent L-Wnt3A cells were split 1:10 into L-Wnt3A conditioning medium. The cells were grown 4 days at 37 C. The supernatant was harvested , filter sterilized and stored as batch 1.Fresh conditioning medium was added to the cells and cultured for another 3 days. The medium was harvested, filter sterilized and mixed 1:1 with batch 1.<br>
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<img src='https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/600px-Human_H9.png'><br>
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Figure 2: CAGE expression of marker genes in TPM.<br>
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