Keratinocyte Progenitor Research Articles

The lncRNA SNHG26 drives the inflammatory-to-proliferative state transition of keratinocyte progenitor cells during wound healing

The cell transition from an inflammatory phase to a subsequent proliferative phase is crucial for wound healing, yet the driving mechanism remains unclear. By profiling lncRNA expression changes during human skin wound healing and screening lncRNA functions, we identify SNHG26 as a pivotal regulator in keratinocyte progenitors underpinning this phase transition. Snhg26-deficient mice exhibit impaired wound repair characterized by delayed re-epithelization accompanied by exacerbated inflammation. Single-cell transcriptome analysis combined with gain-of-function and loss-of-function of SNHG26 in vitro and ex vivo reveals its specific role in facilitating inflammatory-to-proliferative state transition of keratinocyte progenitors. A mechanistic study unravels that SNHG26 interacts with and relocates the transcription factor ILF2 from inflammatory genomic loci, such as JUN, IL6, IL8, and CCL20, to the genomic locus of LAMB3. Collectively, our findings suggest that lncRNAs play cardinal roles in expediting tissue repair and regeneration and may constitute an invaluable reservoir of therapeutic targets in reparative medicine.

Antimicrobial Peptide Reduces Cytotoxicity and Inflammation in Canine Epidermal Keratinocyte Progenitor Cells Induced by Pseudomonas aeruginosa Infection.

The direct effects and antimicrobial activity of synthetic antimicrobial peptides (AMPs) obtained from dogs, including cBD, cBD103, and cCath, against P. aeruginosa wild-type strain PAO1 and canine keratinocytes were analyzed. Antibacterial effects on planktonic bacteria were assessed by determining the minimum bactericidal concentrations (MBCs) of AMPs and by a time-kill assay. Antibiofilm effects were assessed using the microtiter plate assay. We also evaluated the effects of AMPs on cell cytotoxicity and host immune response induced by stimulating canine epidermal keratinocyte progenitor (CPEK) cells with PAO1 and its LPS. cBD, cBD103, and cCath all exhibited dose-dependent antimicrobial and antibiofilm effects. In particular, 25 μg/mL cBD103 showed rapid bactericidal activity within 60 min and inhibited biofilm formation. In addition, pretreatment with cBD103 (25 µg/mL) and cCath (50 µg/mL) 1 h before stimulation significantly reduced the cytotoxicity of the CPEK cells by PAO1 and LPS-induced IL-6 and TNF-a expressions. cBD had little effect on the response to PAO1 and LPS in the cells. These results indicate the therapeutic potential of AMPs in P. aeruginosa skin infections. However, further studies on the mechanism of action of AMPs in keratinocytes and clinical trials are needed.

Abstract 1799: Regulation of epidermal stem cell proliferation and adhesion by ACSS2

Abstract Acetyl-CoA Synthetase Short-Chain Family Member 2 (ACSS2) is an enzyme that converts acetate into acetyl-CoA, a crucial metabolite required for protein acetylation and lipogenesis. Acetyl-CoA is membrane-impermeable and must be synthesized in the compartment in which it will be used. Among ACSS enzymes, ACSS2 alone is found in the nucleocytoplasmic compartment rather than the mitochondria. This enzyme shows dynamic localization dependent on cell nutrient status, with nutrient stress promoting nuclear localization where ACSS2 activity promotes cell growth and survival. Emerging evidence suggests ACSS2 may also play roles in promoting metastasis, and has been proposed as a potential therapeutic target. However, the role of ACSS2 in controlling normal tissue homeostasis is understudied. Addressing this knowledge gap is critical to ensure safety and efficacy of cancer therapeutics. As 80% to 90% of human cancers originate from epithelial tissues, we have leveraged human epidermal stem cells to further interrogate the role of ACSS2. As progenitor keratinocytes differentiate, they detach from the basal lamina and migrate from the metabolically privileged basal layer to the more challenging environment at the surface of the epidermis. Using shRNA-mediated knockdown, we first found ACSS2 knockdown dramatically decreases progenitor keratinocyte clonogenicity, abolishing proliferative capacity. Analysis of RNA sequencing data following ACSS2 knockdown with five constructs showed significant increases in expression of genes related to cell adhesion, extracellular matrix organization, collagen catabolism, and cell migration, in addition to keratinocyte differentiation. Downregulated genes were associated with cell division, DNA repair, and DNA replication. Live cell imaging revealed knockdown cells show striking differences in cell adhesion compared to control. These results indicate a role for ACSS2 in regulating keratinocyte proliferation and adhesion during the maintenance of epidermal homeostasis. Citation Format: Nicholas K. Chamberlain, Xiaomin Bao. Regulation of epidermal stem cell proliferation and adhesion by ACSS2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1799.

Abstract 7070: MEK-ERK signaling maintains epidermal proliferation through H2A.Z deposition

Abstract Recent studies have connected dysregulation of H2A.Z deposition to glioblastoma as well as lung, pancreatic, and metastatic breast cancers. The H2AZ1 and H2AZ2 isoforms, collectively known as H2A.Z, are histone variants that regulate gene expression, stem cell identity, and differentiation during development and in homeostatic tissue. Most tumors originate from epithelial tissues. We utilize skin epidermis, an accessible model of epithelial tissue, to understand the regulatory mechanisms controlling proliferation and differentiation. Primary human epidermal keratinocytes retain their abilities to proliferate, differentiate, and stratify in vitro. First, we observed that progenitor keratinocytes express high levels of H2A.Z mRNA and protein, and that H2A.Z levels significantly and progressively decrease during four days of calcium-induced differentiation. Using ChIP-sequencing, we find a significant, genome-wide decrease in H2A.Z chromatin occupancy in differentiated keratinocytes. Therefore, we hypothesized that H2A.Z is critical for progenitor function. Knock down of either H2AZ1 or H2AZ2 significantly impaired keratinocyte proliferation in holoclone assays. Furthermore, H2AZ1 or H2AZ2 knockdown inhibited epidermal stratification in organotypic culture. Canonically, two chromatin remodeling complexes, SRCAP and EP400, deposit H2A.Z into nucleosomes. While both SRCAP and EP400 knockdown diminish keratinocyte proliferation, only SRCAP knockdown decreases H2A.Z deposition in epidermal keratinocytes. Finally, we find that MEK and ERK signaling is required to sustain expression of SRCAP, H2AZ1, and H2AZ2. MEK or ERK inhibition is sufficient to significantly decrease H2A.Z occupancy genome-wide in progenitor keratinocytes. Citation Format: Stephenie Droll, Benny Zhang, Celia Xue, Maxwell Levine, Xiaomin Bao. MEK-ERK signaling maintains epidermal proliferation through H2A.Z deposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7070.

Nutritional supplementation of an apple callus extract to target epidermal aging

The global increase in life expectancy has led to a growing elderly population, which places a significant burden on healthcare systems. As a result, there is a growing interest to improve healthspan and promote healthy aging with accessible means, such as nutrition and lifestyle interventions. The skin, the body's largest organ and first line of defense against environmental stressors, is particularly vulnerable to aging due to both intrinsic and extrinsic factors. Various molecular and biochemical changes can drive skin aging, including the depletion of stem cells, which can lead to impaired skin properties. Nutrition plays a crucial role in skin aging, and dietary supplements could be a viable intervention to support a healthy skin. In this study, a plant callus extract from a specific apple variety (Malus Domestica Borkh), the Uttwiler Spätlauber, was evaluated as a dietary supplement to combat epidermal aging. The extract was found to maintain the function of keratinocyte progenitor cells during aging in vitro. Furthermore, an open-label pilot clinical study involving 31 female volunteers showed that the intake of apple callus extract led to improvements in skin elasticity, density, and firmness after 56 days of supplementation. This study highlights the potential of plant callus extracts as natural dietary supplements to support healthy skin aging. The use of plant callus cultures provides a sustainable and reproducible source of bioactive molecules, which is gaining acceptance in the nutraceutical market. These findings suggest that dietary interventions can play a role in promoting healthy skin aging and contribute to a holistic approach to improving healthspan during the aging process.

1453 LncRNA SNHG26 facilitates inflammatory to proliferative state transition of keratinocyte progenitors during wound healing

1453 LncRNA SNHG26 facilitates inflammatory to proliferative state transition of keratinocyte progenitors during wound healing

AGS3 antagonizes LGN to balance oriented cell divisions and cell fate choices in mammalian epidermis.

Oriented cell divisions balance self-renewal and differentiation in stratified epithelia such as the skin epidermis. During peak epidermal stratification, the distribution of division angles among basal keratinocyte progenitors is bimodal, with planar and perpendicular divisions driving symmetric and asymmetric daughter cell fates, respectively. An apically restricted, evolutionarily conserved spindle orientation complex that includes the scaffolding protein LGN/Pins/Gpsm2 plays a central role in promoting perpendicular divisions and stratification, but why only a subset of cell polarize LGN is not known. Here, we demonstrate that the LGN paralog, AGS3/Gpsm1, is a novel negative regulator of LGN and inhibits perpendicular divisions. Static and ex vivo live imaging reveal that AGS3 overexpression displaces LGN from the apical cortex and increases planar orientations, while AGS3 loss prolongs cortical LGN localization and leads to a perpendicular orientation bias. Genetic epistasis experiments in double mutants confirm that AGS3 operates through LGN. Finally, clonal lineage tracing shows that LGN and AGS3 promote asymmetric and symmetric fates, respectively, while also influencing differentiation through delamination. Collectively, these studies shed new light on how spindle orientation influences epidermal stratification.

Revisiting the significance of keratin expression in complex epithelia.

A large group of keratin genes (n=54 in the human genome) code for intermediate filament (IF)-forming proteins and show differential regulation in epithelial cells and tissues. Keratin expression can be highly informative about the type of epithelial tissue, differentiation status of constituent cells and biological context (e.g. normal versus diseased settings). The foundational principles underlying the use of keratin expression to gain insight about epithelial cells and tissues primarily originated in pioneering studies conducted in the 1980s. The recent emergence of single cell transcriptomics provides an opportunity to revisit these principles and gain new insight into epithelial biology. Re-analysis of single-cell RNAseq data collected from human and mouse skin has confirmed long-held views regarding the quantitative importance and pairwise regulation of specific keratin genes in keratinocytes of surface epithelia. Furthermore, such analyses confirm and extend the notion that changes in keratin gene expression occur gradually as progenitor keratinocytes commit to and undergo differentiation, and challenge the prevailing assumption that specific keratin combinations reflect a mitotic versus a post-mitotic differentiating state. Our findings provide a blueprint for similar analyses in other tissues, and warrant a more nuanced approach in the use of keratin genes as biomarkers in epithelia.

459 Regulation of the keratinocyte progenitor to differentiation switch by alternative mRNA splicing

Alternative splicing (AS) of messenger RNAs diversifies gene function by generating protein isoforms with different biological properties. Over 95% of human genes are alternatively spliced, but the functional consequences of most AS events are still not known. Here, using long-read RNA sequencing, we identified 5,826 AS events associated with human epidermal differentiation (percent spliced index >10%, FDR <0.01). AS events were enriched in genes associated with histone modification and phosphorylation (p < 0.001). The most common type of AS was cassette exon splicing, in which an intervening exon between two other exons is included or skipped in the mRNA sequence. We functionally characterized a cassette exon in mitogen-activated protein kinase (MAP3K7), which had not been previously studied in human epidermis. Basal epidermal progenitors preferentially express a shorter MAP3K7 isoform but switch to the longer isoform in suprabasal, differentiated epidermis with inclusion of an additional 81 bp exon. Using isoform-specific knockdown and overexpression, we demonstrate that inclusion of a single exon in MAP3K7 is necessary and sufficient to transition human epidermal keratinocytes from a replicating progenitor state with high clonogenic capacity into a post-mitotic differentiated state. We also performed binding motif analysis of sequences adjacent to epidermal AS events which identified 26 RNA binding proteins predicted to mediate mRNA splicing in epidermal keratinocytes, including ten with previously unstudied roles in the skin. In summary, our study defines the repertoire of alternative mRNA splicing events in the human epidermis and demonstrates the potential of this resource to identify functional AS events and splicing RBPs that regulate epidermal differentiation.

453 H2AZ1 and H2AZ2 regulate divergent programs in epidermal progenitor maintenance

Collectively known as H2A.Z, the H2AZ1 and H2AZ2 isoforms differ by only three amino acids. H2AZ2 emerged in vertebrates, and both histone variants play key roles in modulating mammalian gene expression. However, the regulatory role of H2A.Z in epidermal homeostasis remains unclear. We demonstrate that progenitor keratinocytes express the highest amount of H2A.Z at both the mRNA and protein levels, and that H2A.Z expression decreases during differentiation (p < 0.0001). Knocking down either H2AZ1 or H2AZ2 with four shRNAs per target suppresses keratinocyte proliferation in the holoclone assay (p < 0.0001) and impairs progenitor regeneration in organotypic culture (p < 0.0001).

Melanocyte progenitor cells reside in human subcutaneous adipose tissue.

Based on the assumption that some progenitor cells in an organ might reside in neighboring adipose tissue, we investigated whether melanocyte progenitor cells reside in human subcutaneous adipose tissue. First, we examined the expression of human melanoma black 45 (HMB45) and microphthalmia-associated transcription factor (MITF) in undifferentiated adipose-derived stem cells (ADSCs) by immunostaining, RT-PCR, and western blotting. These two markers were detected in undifferentiated ADSCs, and their expression levels were increased in differentiated ADSCs in melanocyte-specific culture medium. Other melanocytic markers (Melan A, MATP, Mel2, Mel EM, tyrosinase, KIT, and PAX3) were also detected at variable levels in undifferentiated ADSCs, and the expression of some markers was increased during differentiation into the melanocyte lineage. We further showed that ADSCs differentiated in melanocyte-specific culture medium localized in the basal layer and expressed tyrosinase and HMB45 in a 3D epidermal culture system. Melanin deposits were also induced by ultraviolet-light-B (UVB) irradiation. These results demonstrate that melanocyte progenitor cells reside in human subcutaneous adipose tissue and that these cells might have the potential to differentiate into mature melanocytes. Melanocyte and keratinocyte progenitors residing in human subcutaneous tissue can be used for the treatment of skin diseases and skin rejuvenation in the future.

High proliferation and delamination during skin epidermal stratification

The development of complex stratified epithelial barriers in mammals is initiated from single-layered epithelia. How stratification is initiated and fueled are still open questions. Previous studies on skin epidermal stratification suggested a central role for perpendicular/asymmetric cell division orientation of the basal keratinocyte progenitors. Here, we use centrosomes, that organize the mitotic spindle, to test whether cell division orientation and stratification are linked. Genetically ablating centrosomes from the developing epidermis leads to the activation of the p53-, 53BP1- and USP28-dependent mitotic surveillance pathway causing a thinner epidermis and hair follicle arrest. The centrosome/p53-double mutant keratinocyte progenitors significantly alter their division orientation in the later stages without majorly affecting epidermal differentiation. Together with time-lapse imaging and tissue growth dynamics measurements, the data suggest that the first and major phase of epidermal development is boosted by high proliferation rates in both basal and suprabasally-committed keratinocytes as well as cell delamination, whereas the second phase maybe uncoupled from the division orientation of the basal progenitors. The data provide insights for tissue homeostasis and hyperproliferative diseases that may recapitulate developmental programs.

146 A two-phase model of epidermal stratification: Lessons from centrosomes

The stratified skin epidermal barrier in mammals develops from a single-layered epithelium. The mechanisms initiating epidermal stratification and how the process is fueled are still not well-understood. Previous studies proposed a central role for perpendicular/asymmetric cell division orientation of the basal keratinocyte progenitors in epidermal stratification. Here, we used centrosomes, that organize the mitotic spindle, to test whether cell division orientation and stratification are linked. Genetic ablation of centrosomes from the developing epidermis led to the activation of the p53-dependent mitotic surveillance pathway resulting in reduced epidermal thickness and hair follicle arrest. Double mutant keratinocyte progenitors lacking both centrosomes and p53 rescued the epidermal phenotypes. Importantly, the mutant progenitors significantly altered their division orientation in the later developmental stages without affecting stratification or differentiation. Based on time-lapse imaging and analysis of tissue growth dynamics, our data suggested a two-phase model of epidermal stratification. The first and major phase of epidermal stratification is characterized by highly proliferating basal and suprabasal transit-amplifying cells as well as cell delamination, while the second phase may be uncoupled from the division orientation of the basal progenitors. The data provide insights for tissue homeostasis and hyperproliferative diseases that may recapitulate developmental programs.

The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation.

While the mechanisms by which chemical signals control cell fate have been well studied, the impact of mechanical inputs on cell fate decisions is not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through linker of nucleoskeleton and cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

Melanin Distribution in Human Skin: Influence of Cytoskeletal, Polarity, and Centrosome-Related Machinery of Stratum basale Keratinocytes.

Melanin granules cluster within supra-nuclear caps in basal keratinocytes (KCs) of the human epidermis, where they protect KC genomic DNA against ultraviolet radiation (UVR) damage. While much is known about melanogenesis in melanocytes (MCs) and a moderate amount about melanin transfer from MC to KC, we know little about the fate of melanin once inside KCs. We recently reported that melanin fate in progenitor KCs is regulated by rare asymmetric organelle movement during mitosis. Here, we explore the role of actin, microtubules, and centrosome-associated machinery in distributing melanin within KCs. Short-term cultures of human skin explants were treated with cytochalasin-B and nocodazole to target actin filaments and microtubules, respectively. Treatment effects on melanin distribution were assessed by the Warthin–Starry stain, on centrosome-associated proteins by immunofluorescence microscopy, and on co-localisation with melanin granules by brightfield microscopy. Cytochalasin-B treatment disassembled supra-nuclear melanin caps, while nocodazole treatment moved melanin from the apical to basal KC domain. Centrosome and centriolar satellite-associated proteins showed a high degree of co-localisation with melanin. Thus, once melanin granules are transferred to KCs, their preferred apical distribution appears to be facilitated by coordinated movement of centrosomes and centriolar satellites. This mechanism may control melanin’s strategic position within UVR-exposed KCs.

Comparative Analysis of Cx31 and Cx43 in Differentiation-Competent Rodent Keratinocytes

When considering connexin expression and regulation, the epidermis of the skin is one of the most complex tissues found in mammals even though it largely contains a single cell type, the keratinocyte. In the rodent epidermis, up to 9 connexin family members have been detected at the mRNA level. Many of these connexins are temporally and spatially regulated in coordination with keratinocyte progenitor cell differentiation and migration from the stratum basale to form the stratum spinosum and stratum granulosum layers before finally forming the stratum corneum. Cx43 is the principal connexin found in basal keratinocytes and to a lesser degree found in keratinocytes that have begun to differentiate where Cx26, Cx30 and Cx31 become prevalent. Here we show that the CRISPR-Cas9 ablation of Cx43 reduces overall gap junction coupling in monolayer cultures of rat epidermal keratinocytes (REKs) and dysregulates the differentiation of REKs when grown in organotypic cultures. Natively found in differentiated keratinocytes, Cx31 readily assembles into gap junctions when expressed in REKs where it can extensively co-assemble into the same gap junctions with co-expressed Cx30. Time-lapse imaging indicated that many Cx31 gap junctions are mobile within the plasma membrane undergoing both fusion and fission events. Finally, the persistence of pre-existing Cx31 gap junctions in the presence of the protein trafficking blocker, brefeldin A, is longer than that found for Cx43 gap junctions indicating that it has a distinctly different life expectancy in REKs. Collectively, this study highlights the importance of Cx43 in rodent keratinocyte differentiation and suggests that Cx31 acquires life-cycle properties that are distinct from Cx43.

Deriving Keratinocyte Progenitor Cells and Keratinocytes from Human-Induced Pluripotent Stem Cells.

Skin or hair loss (alopecia) may occur due to a wide variety of causes ranging from trauma to pathological processes including acquired or congenital causes. It would be ideal to replace them with immunologically compatible cells to avoid potentially exacerbating the condition. Deriving the replacement cells from human-induced pluripotent stem cells (hiPSCs) allows for sufficient scale up and using hiPSCs as the choice of human pluripotent stem cells (hPSC) will ensure immunocompatibility. Here we offer a protocol for differentiating hiPSCs into keratinocyte progenitor cells (KPC) and keratinocytes employing all-trans retinoic acid (ATRA) and L-ascorbic acid, (L-AA), bone morphogenic protein-4 (BMP4), and epidermal growth factor (EGF). We observed that the hiPSC-derived KPCs express the same panel of markers as primary hair follicle bulge stem cells (HFBSCs), including CD200, integrin α-6 (ITGA6), integrin β-1 (ITGB1), the transcription factor P63, keratin 15 (KRT15), and keratin 19 (KRT19). If permitted to differentiate further, the hiPSC-derived KPC lose CD200 expression and rather come to express keratin 14 (KRT14) indicating emergence of more mature terminally-differentiated keratinocytes. The HFBSCs are transplantable for hair follicle (HF) restoration, and the keratinocytes may be transplantable for therapy for large burns or ulcers. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Reprogramming of normal human skin fibroblasts into normal hiPSCs using episomal DNA cocktail Basic Protocol 2: Differentiation of hiPSCs into KPCs and keratinocytes Alternate Protocol 2: EBS formation protocol using AggreWell™ plates (Antonchuk, 2013) Support Protocol 1: Passage hiPSC-KPC Support Protocol 2: Immunocytochemistry (ICC) Support Protocol 3: Immunofluorescence staining of cells for flow cytometry (FC).

Molecular Mapping of Hydrogen Sulfide Targets in Normal Human Keratinocytes.

Although sulfur-rich thermal waters have ancestrally been used in the context of dermatological conditions, a global mapping of the molecular effects exerted by H2S on human keratinocytes is still lacking. To fill this knowledge gap, we subjected cultured human keratinocytes to distinct amounts of the non-gaseous hydrogen sulfur donor NaHS. We first checked that H2S accumulated in the cytoplasm of keratinocytes under our experimental conditions andused a combination of proteomics, genomics and biochemical approaches to unravel functionally relevant H2S targets in human keratinocytes. We found that the identified targets fall into two main categories: (i) the oxidative stress response molecules superoxide dismutase 2 (SOD2), NAD(P)H quinone dehydrogenase 1 (NQO1) and culin 3 (CUL3) and (ii) the chemokines interleukin-8 (IL-8) and CXCL2. Interestingly, NaHS also stimulated the caspase-1 inflammasome pathway, leading to increased secretion of the pro-inflammatory molecule interleukin-18 (IL-18). Interestingly, the secretion of interleukin-1 beta (IL-1β) was only modestly impacted by NaHS exposure despite a significant accumulation of IL-1β pro-form. Finally, we observed that NaHS significantly hampered the growth of human keratinocyte progenitors and stem cells cultured under clonogenic conditions or as epidermal cell sheets. We conclude that H2S exerts specific molecular effects on normal human keratinocytes.

766 Keratinocyte differentiation is coupled to mechanical cues through the LINC complex

Skin tissue is resilient to mechanical forces, but also can respond to prolonged mechanical forces. For example, tissue expanders are used to generate more skin for breast reconstruction surgery. At the cellular level, multiple mechanical forces are exerted on epidermal keratinocytes. Progenitor keratinocytes, unlike differentiated keratinocytes, have both cell-cell adhesions and cell-extracellular matrix (ECM) adhesions that keep progenitor keratinocytes attached to the basement membrane. During keratinocyte differentiation, progenitor keratinocytes lose cell-ECM adhesions and detach from the basement membrane, but the exact mechanism of how this occurs is not fully understood. External forces must be communicated from the plasma membrane at the outside of the cell to the nucleus, and the Linker of Nucleoskeletal and Cytoskeletal (LINC) complex is poised on the nuclear membrane to sense these mechanical forces. We were interested in determining if there is tension on the LINC complex in keratinocytes and if this tension influences the function of epidermal keratinocytes. Tension on the LINC complex was measured using a FRET-based Nesprin tension sensor, which showed that keratinocytes undergoing differentiation have less tension on the LINC complex compared to progenitor keratinocytes. To investigate the functional consequences of tension on the LINC complex, mice lacking LINC complex components SUN1 and SUN2 proteins were generated. Multiple lines of evidence indicate that Sun1/2 null keratinocytes undergo precocious differentiation both in culture and in vivo. In conclusion, this work demonstrates that mechanical tension on the LINC complex maintains keratinocytes in a progenitor state and identifies tension on the LINC complex as a new regulator of keratinocyte differentiation.

Influence of cryostorage on stem cell population in human keratinocytes

Background & Aim During many years living skin equivalent (LSE) development has been an actual topic in regenerative medicine. Immature phenotype and high clonogenic potential of keratinocytes is related to the ability of the cells to reconstruct a pluristratified epidermis demonstrating an optimal balance between proliferation and differentiation. Cryostorage becomes a crucial stage of LSE development which potentially can influence keratinocyte stem cell (KSC) population. Towards this end, we wanted to evaluate cell characteristics before and after cryostorage. Methods, Results & Conclusion RNA expression of hemidesmosomal genes (integrins α6, β4 and β1), specific for epidermal basal layer, was examined by rtPCR analysis of keratinocyte primary culture before (p0) and after cryostorage (p1 – 1 and 7 day). Cell percentage variation with KSC phenotype (CD71-/integrin β4+) before (p0) and after cryostorage (1day) was measured by flow cytometry. Protein expression of integrin α6 before (p0) and after cryostorage (1 and 3 day) was measured by Western blot. Keratinocyte clonogenic potential was assessed by Colony Forming Efficiency test (CFE%) using standard protocol (J.Rheinwald, H. Green) before cryostorage (p2) and after thawing (p1-first passage after thawing and p2- second passage after 3 or 10 days of p1 cultivation). At the first day after thawing 12.5% of all live cells attached to plastic. Among them there was a decrease in RNA expression of α6 and β1 integrins; while increase in RNA expression of α6 integrin was observed on day 7. Wherein protein content of integrin α6 was the same before and after (1 day) thawing, but decreased to day 3. Besides, we found a rising rate of cell percentage with KSC phenotype after thawing. In addition, we observed an increase of keratinocyte clonogenic potential after thawing (CFE%): holoclones (d〉400μm) number in p2 after 10 days post-thawing keratinocyte cultivation was significantly higher compared to that before cryostorage. In contrast, if p1 was cultured for 3 days after thawing only, keratinocytes’ clonogenic potential in p2 was lower than prior to preservation. CFE test if performed immediately after thawing revealed absence of colonies. Presumably, hemidesmosomal genes expression transiently decreases in keratinocytes after cryostorage. We suggest that selection of progenitor keratinocytes with high density of integrin α6 molecules on their surface occurs. Funded by IDB RAS government program.