tissue: dermis gender: female age: 16y at reprogramming
Treatment protocol
For MSC induction, embryo bodies were formed and kept for 2 days floating inhiPSC medium consisting of DMEM/Ham’s F12 medium (Sigma) containing 20% Knockout Serum Replacement (Life Technologies), 2 mM L-glutamine (Sigma), 1×10-4 M nonessential amino acids (Sigma), 1×10-4 M 2-mercaptoethanol (Sigma), 4 ng/ml FGF2 (Wako, Osaka, Japan), 0.5% penicillin and streptomycin (Life Technologies). Subsequently, they were cultured in Stempro MSC-SFM CTS (Life Technologies) on a humanised substrate, CELLstart CTS (Life Technologies) and cultured for a further 9-11 days as described in growth protocol. For sorting, hiPSC-MSC were detached, dissociated and incubated with mouse anti-human CD271 phycoerythrin-conjugated (Biolegend, San Diego, CA) and anti-human CD90 allophycocyanin-conjugated monoclonal antibodies (Biolegend) for 30 minutes. Single staining by each antibody was used as controls. Flow cytometric sorting was then performed on MoFlo XDP (Beckman Coulter, Brea, CA). The resultant cells were subject to dermal papilla induction culture as described in growth protocol.
Growth protocol
Freshly microdissected human DP cells were cultured in PromoCell Follicle Papilla Cell Growth Medium (PromoCell, Heiderberg, Germany). hiPSCs-MSCs were cultured in Stempro MSC-SFM CTS (Life Technologies, Carlsbad, CA) supplemented with bFGF on a humanised substrate, CELLstart CTS (Life Technologies). After sorting, CD271+CD90+hiPSC-MSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 0.01 mM all-trans retinoic acid (Sigma, St. louis, MO) (day 0-4). On day 4, induction medium was then changed to DMEM with 10%FBS, 20 ng/ ml bFGF (Peprotech, Rocky Hill, NJ), 200 ng/ ml human recombinant BMP2 (R&D Systems, Minneapolis, MN) and 1 μM 6-bromoindirubin-3'-oxime (Sigma) (day 4-9).
Extracted molecule
total RNA
Extraction protocol
Total RNA was harvested and purified using the RNeasy Mini (Qiagen, Hilden, Germany) following manufacutures instrcution. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1ug RNA using the Low Input Quick Amp Labelling Kit, one-color (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
0.6 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human Gene Expression 8x60K v2 microarray for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B)
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. These data were normalized by 75th percentile normalisation by GeneSpring GX with default condition. The probes less than 100 in all six samples were filtered out. The full set of normalized data is available on the series record.
Gene expression data of human dermal pilla cells, human induced pluripotent stem cell-derived CD271+CD90+ mesenchymal stem cells before and after the induction toward dermal papilla cells
