Live cell imaging is an established method that can be used to study keratinocyte division kinetics in vitro. To minimize the influence of the culture environment, unpassaged human keratinocytes can be utilized. To study the kinetics of single cells, keratinocytes are grown at clonal density and divisions are recorded utilizing time lapse photography. From this, lineage trees are constructed, allowing determination of cell cycle duration and division fates of individual keratinocytes. Here we describe a method for the culture of human keratinocytes for live cell imaging, methods for analysis of the photography, and how to assess proliferation, differentiation, and cell cycle duration.

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.

Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. Historically, keratinocyte division behavior was investigated via methods such as clonal analysis, immunostaining, and cell cycle analysis. None of these methods allow for the analysis of keratinocyte behavior at the single-cell level in real time. Over the past decade, groups have utilized live cell imaging to identify keratinocyte stem cells and committed progenitors without the need for labeling. Differences have been identified in each respective group's division behavior, rate of terminal differentiation, and cell cycle duration. Here, a method for keratinocyte live cell imaging with time-lapse photography and its analysis is described. Utilizing unpassaged keratinocytes is recommended for this method to most closely mimic in vivo behavior. Live cell imaging provides a unique ability to study stem cell and committed progenitor behavior at the single cell level and to determine division fates, cell cycle duration, as well as other proliferation metrics.

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in vitro. Live-cell imaging has allowed for significant advances in the ability to identify differences between keratinocyte stem cells and committed progenitor behavior. Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. This protocol has been developed to culture keratinocytes at low seeding density, enabling relatively long-term time-lapse photography and monitoring of individual cell behavior. Passage 0 keratinocytes are grown at clonal density, and time-lapse photography allows documentation of individual cell divisions and the time of their occurrence. For maximum biological relevance, freshly isolated human keratinocytes are placed in vitro. This approach focuses on proliferation. However, this protocol can be adapted for use in other live cell imaging applications measuring individual cell behavior, such as measuring cell migration, wound healing, and motility.

Loss of sensory innervation delays wound healing and administration of the neuropeptide substance P improves re-epithelialization. Keratinocyte hyperproliferation post-wounding may result from symmetric stem cell (SC) self-renewal, asymmetric SC self-renewal, committed progenitor divisions, or a combination of these. However, the effects of sensory denervation and of neuropeptides on SC proliferation are not known. Here we show that early after wounding both asymmetric and symmetric SC self-renewal increase, without significant committed progenitor (CP) activation. Decreased sensory innervation is associated with a decrease in both SC and CP proliferation. Based on previous work showing that substance P is decreased in capsaicin-treated mice and improves wound healing in normal skin, we examined the effects of substance P on SC and CP proliferation during wound healing. Substance P restored asymmetric SC proliferation in skin with decreased sensory innervation, both at baseline and following wounding. Epidermis with decreased sensory innervation was severely thinned. Consistent with this, substance P-induced asymmetric SC proliferation resulted in increased stratification in skin with both normal and decreased innervation. Lapatinib prevented the substance P-induced increase in asymmetric SC divisions in murine epidermis, as well as the increase in epidermal stratification, suggesting that asymmetric SC divisions are required for epidermal stratification.

Background A traditional view is that stem cells (SCs) divide slowly. Meanwhile, both embryonic and pluripotent SCs display a shorter cell cycle duration (CCD) in comparison to more committed progenitors (CPs). Methods We examined the in vitro proliferation and cycling behavior of somatic adult human cells using live cell imaging of passage zero keratinocytes and single-cell RNA sequencing. Results We found two populations of keratinocytes: those with short CCD and protracted near exponential growth, and those with long CCD and terminal differentiation. Applying the ergodic principle, the comparative numbers of cycling cells in S phase in an enriched population of SCs confirmed a shorter CCD than CPs. Further, analysis of single-cell RNA sequencing of cycling adult human keratinocyte SCs and CPs indicated a shortening of both G1 and G2M phases in the SC. Conclusions Contrary to the pervasive paradigm, SCs progress through cell cycle more quickly than more differentiated dividing CPs. Thus, somatic human adult keratinocyte SCs may divide infrequently, but divide rapidly when they divide. Additionally, it was found that SC-like proliferation persisted in vitro.

Whether epidermal stem cells (SCs) maintain their phenotype in vitro remains largely unknown. To determine whether adult human SCs maintain their self-renewal behavior in vitro, we observed adult keratinocytes in vitro. We studied live cell imaging of freshly obtained normal adult human keratinocytes from surgical discards over 11-16 days. We then tracked each colony and defined cells that divided as P (proliferating) and cells that did not divide (in 48h) as D (differentiated). The three outcomes of divisions were classified as PP, PD, and DD. We were able to define SCs, as was done for neonatal keratinocytes (Roshan et al, 2016), by 100% PP divisions over the first 3 generations. SCs formed significantly larger colonies, as expected. Committed progenitor colonies mostly terminally differentiated (75%) during the observation period, whereas no SC colonies did. Over the period of observation, we found that in SC colonies PP divisions dominated (82.8%±6.5), even at 10-14 generations, while in committed progenitor colonies PP divisions were only 30.6%±4.0 of divisions (p<0.0001). Our relatively long observation (up to 16 days) allowed us to determine that SCs maintained a high proportion (>70%) of PP divisions over time. In conclusion, adult human epidermal SCs preserved their self-renewal behavior in culture. Further studies are needed to determine further properties of keratinocyte SCs in vitro.

Psoriasis is an immune-mediated skin condition characterized by hyperproliferation of keratinocytes. Psoriasis stem cells (SCs) have previously been shown to exhibit increased asymmetric SC self-renewal without a change in SC number. To better understand the genes modulating SC and committed progenitor cell (CP) proliferation in psoriasis, we reexamined the Cheng et al. 2018 scRNA sequencing data comparing truncal samples (n=3) from psoriatic and normal human epidermis. Focusing on the mitotic cell types, we utilized an unbiased subclustering approach with pseudotime analysis to isolate four clusters corresponding to putative SCs and CPs. All cells expressing differentiated markers (IVL and FLG) or markers for melanocytes and immune cells were discarded in order to isolate mitotic keratinocytes in the basal and lower spinous layers. We found that the number of CPs is increased eight-fold in psoriasis with no detected change in the number of SCs in the basal layer. As expected, markers for cell proliferation were differentially upregulated in psoriasis for all mitotic keratinocytes. However, we also identify genes highly expressed in specific mitotic psoriasis clusters that highlight their possible role in disease pathogenesis including, POUF51 (upregulated in SCs), a regulator of stem cell maintenance and HES1 (upregulated in CPs), a Notch target gene involved in keratinocyte asymmetric SC division. Understanding the basis for SC and CP hyperproliferation in psoriasis may lead to novel therapeutic targets for the skin manifestations of psoriasis in patients with mild to moderate disease.

Neuropeptides improve wound healing, but little is known about how they affect stem cells (SC), committed progenitors (CP), and relevant signaling pathways to increase proliferation. Mice with Capsaicin-induced decreased sensory innervation (DSI) were treated with Substance P (SP). 6mm wounds were analyzed at 4 days post- wounding (in the most proliferative phase of healing). Keratinocyte division analysis was studied using Numb and keratin 1 expression. In Capsaicin vs. vehicle treated mice, wound healing was delayed, (24.8±0.3 days vs. 14.5±0.3 days (n= 8,P

Little is known about the kinetics of hyperproliferation in wound healing, the role of stem cells (SC) and transit amplifying cells (committed progenitors - CP), and relevant SC signaling pathways. To study SC and CP proliferation during wound healing, 6mm wounds were made on the flanks of mice and cell divisions assessed 4d post-wounding using fluorescence microscopy; in vivo using tubulin expression and in vitro using Numb and keratin 1 expression. In wounded vs. intact skin in vivo there were increased basal, but not suprabasal, divisions, with an increase in both perpendicular (asymmetric self-renewal- ASR) and parallel (symmetric) divisions in in the basal layer (4.9 vs. 1.3 /10 cm P=0.001 and 6.8 vs. 4.6 /10 cm P=0.01, respectively, n=4). In vitro, where ASR and symmetric self-renewal (SSR) SC divisions can be distinguished from symmetric differentiation (SD) of CPs, an increase in both ASR and SSR divisions per 100 cells was seen, (3.3 vs. 2.1, P=0.01 and 0.7 vs. 0.3, P=0.03 - n=4) with no significant change detected in symmetric differentiation (8.5 vs. 8.3, ns), suggesting that the proliferative response early on is confined to SCs. Substance P is important for wound healing and induced epidermal proliferation, improved wound healing, and increased epidermal thickness, as previously shown. Substance P treatment of wounds increased total, ASR, and SD divisions per 100 cells, but not SSR (total 12.9 vs. 9.0, ASR 3.1 vs. 1.9, SSR 0.9 vs. 0.7, and SD 9.1 vs. 6.7, all P<05, except SSR P=.2). In summary, while psoriasis is associated with increased ASR and squamous cell carcinoma with increased SSR, wound healing is associated with increases in both ASR and SSR. Substance P induced improvement of wound healing is associated with increased asymmetric SC self-renewal divisions. Understanding the effects of neuropeptides on SC behavior is important in the search for better wound healing therapies.