The CD90+CD39+ and CD90+CD39- fibroblast subtypes in human hypertrophic scar and normal skin

To explore the functional difference between CD90+CD39+ and CD90+CD39- fibroblasts in human hypertrophic scar and normal skin, the gene expresson microarray was performed on Live CD49f- E-Cadherin- Lin- CD45- CD31- CD90+ CD39+ and Live CD49f- E-Cadherin- Lin- CD45- CD31- CD90+ CD39- cells sorted from suspension disgested from three human hypertrophic scar samples; and Live CD49f- E-Cadherin- Lin- CD45- CD31- CD90+ CD39+ cells sorted from suspension disgested from three human normal skin samples Three 10 cm × 10 cm samples of hypertrophic scar and three 5 cm × 5 cm samples of normal skin were cut into small pieces and put into Dispase II (Gibco, 17105041, 0.3% in DMEM) for 30min at 37℃ to allow the separation of the epidermis. Scar were then minced and digested with a modified protocol, namely, 2.5 mg/mL Collagenase I (Gibco, 17100017), 1 mg/mL Collagenase II (Gibco, 17101015), 1 mg/mL Collagenase IV (Gibco, 17104019), 0.2 mg/mL Hyaluronidase (Sigma, H3884) and 0.1 mg/mL DNase Ⅰ(Sigma, D4263) in DMEM + 2% P/S (Gibco, 15070-063) for 4h shaking in a 37°C waterbath. Acquired cell suspension was filtered, lysed with ACK Lysis Buffer, stained with FVS780- APC-CY7 and Human Fc block. Afterwards, cells were stained with anti-human antibodies for 10min at 4°C: Lineage Cocktail-Pacific Blue (BD, 562722, 1:100), CD45- APC-CY7 (BD, 557833, 1:100), E-Cadherin-BV421 (BD, 743712, 1:100), CD31-PE-CY7 (BD, 563651, 1:100), CD49f/ITGA6-PE (BD, 555736, 1:50), CD90-BV510 (BD, 563070, 1:20), and CD39-PE-CF594 (BD, 563678, 1:100). After negative selection (singlets, FVS780-, CD49f-, E-Cadherin-, Lin-, CD45-, and CD31-), CD90+CD39+ and CD90+CD39- Fb from hypertrophic scar tissue and CD90+CD39+ Fb from normal skin were sorted using BD FACS Aria II. Sorted cells were collected in TRIzol™ Reagent (Invitrogen, Carlsbad, CA, USA) for RNA isolation and subjected to Agilent Human 4x44K Gene Expression Microarrays v2 according to Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology)