397 Sh-oligopeptide-72 ameliorates the proliferative defects of aging keratinocytes

Objectives/Goals: Aged keratinocytes are less proliferative than adult, and aged skin heals more slowly. We examined the proliferation kinetics of aged and adult human keratinocytes. We then tested whether an extrinsic agent, sh-oligopeptide-72, can ameliorate these defects. Methods/Study Population: We used live cell imaging (LCI) to examine the proliferation kinetics of aged (73–92y) and adult (34–49y) passage zero human keratinocytes. We then incubated aged keratinocytes with a peptide, sh-oligopeptide-72 (purported to improve keratinocyte proliferation), or vehicle (PBS). Lineage trees of cell divisions were constructed to determine cell cycle duration and the proliferation/differentiation outcomes of each division. To assess wound healing, cells were isolated from 3 patients, 82–92y, and plated in 2-well culture dishes with inserts. Wells were treated with sh-oligopeptide-72 (100 ng/ml) or vehicle (PBS). At confluence, the insert was removed leaving a well-defined 500 μm gap. The time until 100% closure of the defect was obtained using LCI and the wound healing size tool. Results/Anticipated Results: There was no significant difference in the number of stem cell (SC) colonies between aged and adult keratinocytes. However, aged keratinocytes produced more aged committed progenitor (CP) colonies (P<0.0001). Adult CP, but not stem, colonies were significantly larger than aged (P = 0.0001), and this was associated with earlier terminal differentiation (P = 0.0005). Aged SC and CP colonies exhibited a higher proportion of differentiation divisions, and their cell cycle duration (CCD) was increased. Sh-oligopeptide-72 rescued the increased terminal differentiation as well as decreased the CCD in SC colonies. Sh-oligopeptide decreased the mean closure time of the wound assays (143h vs. 204h, P = 0.04). Discussion/Significance of Impact: Sh-oligopeptide-72 reversed many of the proliferation defects that develop in aged SC colonies. Wound assays show that this results in improved keratinocyte function. These results suggest that the age-related changes in growth dynamics can be modified in response to extrinsic signals in vitro.