Melanocytic differentiation
Human H9 embyronic stem cells differentiated to melanocytes
Time course ID: human_H9
Introduction
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.
Samples
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.
Quality control
Figure 2: CAGE expression of marker genes in TPM.
References
1. Defining the conditions for the generation of melanocytes from human embryonic stem cells.
[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.
2. Embryonic stem cells as a model for studying melanocyte development.
[2] Zabierowski SE, Herlyn M. Methods Mol Biol. 2010;584:301-16.
Beginning of non-public section
Paper outline:
No immediate paper planed.
Bioinformatics collaborators
Emmanuel Dimont and Winston Hide
Piotr Balwierz and Peter Pemberton-Ross
Results
Zenbu configurations and status (COMPLETED)
- <a href="https://fantom5-collaboration.gsc.riken.jp/zenbu/gLyphs/#config=COygWebwCpGoQRErwbLQR">H9 Embryonic Stem cells melanocyte induced</a>
- <a href="https://fantom5-collaboration.gsc.riken.jp/zenbu/gLyphs/#config=5D_TzjfVHRllB6DdxrqnSD;loc=hg19::chr19:36376423..36402712">H9 Embryonic Stem cells melanocyte induced timecourse, October 26th 2012</a>
Expression profiles
- Gene and CAGE cluster expression for the melanocytic differentiation series
MARA based network results
Self-organising maps <img src="ef=" _fck_mw_filename="Melanocytic.pdf" alt="" />
TSS Switching (Switch Engine)
- TSS dynamics plots can be found here:<a href="H9embryoidbody.plots.pdf" class="internal" _fcknotitle="true" _fck_mw_filename="H9embryoidbody.plots.pdf" _fck_mw_type="media" title="H9embryoidbody.plots.pdf">H9embryoidbody.plots.pdf</a> (it is recommended to download these files instead of viewing in browser as they are large in size)
- These figures only show those genes for which Switch Engine has detected TSS switching to occur. For now, only the first and last time points are being compared to define a switch.
- One gene per page, RefSeq and Gene Symbol identifiers on top. Each panel is a separate TSS associated with the gene. TPM expression on y-axis and time on x-axis. Colored points correspond to specific expression, with colors referring to different replicates (key on top). Replicates with a * next to the name are those that have missing data from the currently available release of the DPI clustered and normalized TPM matrix. Such missing data were replaced by imputed values. Black lines are the mean trajectories across replicates. Purple vertical bars correspond to the time points being compared for switch definition. Magnitude of switch is expressed in each panel under TSS identifier with approximate 95% confidence interval in brackets. Note: some confidence intervals may not be accurate or may be missing due to the small sample size. TSS identifiers colored GREEN show an increasing TSS, those colored RED show decreasing TSS, WHITE show no statistically significant change.
- TSS switch defined as the simultaneous presence of one or more increasing, together with one or more decreasing TSSs per gene. At least one increase and at least one decrease has to be statistically significant (alpha approximately 5%). At least one of the two time points being compared has to be greater than 5 tpm. At least 2 replicates have to agree to call an increasing/decreasing trajectory. TSSs have to be at least 250 base pairs away from one another to call a switch.
- A summary file can be found here: <a href="H9embryoidbody.summary.xls" class="internal" _fcknotitle="true" _fck_mw_filename="H9embryoidbody.summary.xls" _fck_mw_type="media" title="H9embryoidbody.summary.xls">H9embryoidbody.summary.xls</a>
- The file contains the following columns: RefSeq ID, Gene Symbol, TSS ID, X{value}: mean tpm expression at {value} time point, Delta: change in tpm expression from first to last time point, FC: fold change in tpm expression from first to last time point, DeltaSum: net change in tpm expression for this gene (summed across all TSS in this gene), Balance: ratio of increasing to decreasing tpm expression TSSs per gene (e.g. a balance value of 1 means that the total increase in tpm expression in increasing TSSs is equal to the total decrease in tpm expression in decreasing TSSs), Chromosome, Strand, Start position of TSS cluster, Stop position of TSS cluster, DBTSS: distance between TSSs relative to NA value.
- A list of genes in which TSS switching occurs can be found here: <a href="H9embryoidbody.genelist.refseq.doc" class="internal" _fcknotitle="true" _fck_mw_filename="H9embryoidbody.genelist.refseq.doc" _fck_mw_type="media" title="H9embryoidbody.genelist.refseq.doc">H9embryoidbody.genelist.refseq.doc</a> and <a href="H9embryoidbody.genelist.symbol.doc" class="internal" _fcknotitle="true" _fck_mw_filename="H9embryoidbody.genelist.symbol.doc" _fck_mw_type="media" title="H9embryoidbody.genelist.symbol.doc">H9embryoidbody.genelist.symbol.doc</a>
- BED file with regions corresponding to TSS clusters involved in TSS switching can be found here: <a href="H9embryoidbody.switchinglist.bed.xls" class="internal" _fcknotitle="true" _fck_mw_filename="H9embryoidbody.switchinglist.bed.xls" _fck_mw_type="media" title="H9embryoidbody.switchinglist.bed.xls">H9embryoidbody.switchinglist.bed.xls</a> (please remove .xls extension after downloading)
Related samples
- ...
Quality control
Isolated RNA samples were tested for the presence of tyrosinase mRNA as control of successful differentiation to melanocytes. Quality control of the hCAGE data have been performed at RIKEN.
ISMARA analysis results
All samples: http://ismara.unibas.ch/timecourses/melanocytes2/ismara_report/index.html
Replicate averaged:
http://ismara.unibas.ch/timecourses/melanocytes2-avgd/averaged_report/index.html
For more information, see <a _fcknotitle="true" href="ISMARA">ISMARA</a>.
