scaned-an-open-source-skin-segmentation-macro-for-semi-automated-cell

SCAnED - An Open-source Skin Segmentation Macro for Semi-automated Cell and Nuclei Detection in Epidermal and Dermal Skin Compartments

The spatial distribution of immune and non-immune cells within tissues and the expression of cell-specific markers provides essential information on cell ...

Multipotent mesenchymal stromal cells (MSCs) are found in a variety of adult tissues including human dermis. These MSCs are morphologically similar to bone marrow-derived MSCs, but are of unclear phenotype. To shed light on the characteristics of human dermal MSCs, this study was designed to identify and isolate dermal MSCs by a specific marker expression profile, and subsequently rate their mesenchymal differentiation potential. Immunohistochemical staining showed that MSC markers CD73/CD90/CD105, as well as CD271 and SSEA-4, are expressed on dermal cells in situ. Flow cytometric analysis revealed a phenotype similar to bone marrow-derived MSCs. Human dermal cells isolated by plastic adherence had a lower differentiation capacity as compared with bone marrow-derived MSCs. To distinguish dermal MSCs from differentiated fibroblasts, we immunoselected CD271(+) and SSEA-4(+) cells from adherent dermal cells and investigated their mesenchymal differentiation capacity. This revealed that cells with increased adipogenic, osteogenic, and chondrogenic potential were enriched in the dermal CD271(+) population. The differentiation potential of dermal SSEA-4(+) cells, in contrast, appeared to be limited to adipogenesis. These results indicate that specific cell populations with variable mesenchymal differentiation potential can be isolated from human dermis. Moreover, we identified three different subsets of dermal mesenchymal progenitor cells.

Human dermis harbors distinct mesenchymal stromal cell subsets.

Multipotenten Mesenchymale Stromale Zellen (MSCs) befinden sich in einer Vielzahl von erwachsenen Geweben, einschlie&szlig;lich menschliche Lederhaut. Diese MSCs sind Knochenmark abgeleitete MSCs morphologisch &auml;hnlich, jedoch von unklar Ph&auml;notyp. Um Aufschluss &uuml;ber die Eigenschaften des menschlichen dermalen MSCs dieser Studie soll identifizieren und isolieren dermal MSCs durch ein spezifischer Marker Ausdruck Profil und anschlie&szlig;end bewerten ihre Mesenchymale Differenzierung Potenzial. Immunhistochemische F&auml;rbung zeigte, dass MSC Marker CD73/CD90/CD105, sowie CD271 und SSEA-4, in-situ dermal Zellen exprimiert werden. Flow Cytometric Analyse ergab, dass ein Ph&auml;notyp &auml;hnlich Knochenmark gewonnenen MSCs. Mensch dermal Zellen durch Einhaltung der Kunststoff isoliert eine geringere Differenzierung Speicherkapazit&auml;t im Vergleich zu MSCs Knochenmark abgeleitet hatte. Dermale unterscheiden MSCs aus Fibroblasten, wir Immunoselected CD271(+) und SSEA-4(+) Zellen von anhaftenden dermal Zellen differenziert und untersucht ihre Mesenchymale Differenzierung-Kapazit&auml;t. Dies offenbart, dass Zellen mit erh&ouml;hter Adipogenic, osteogenen, und faserreiche Potenzial in der dermalen CD271(+) Bev&ouml;lkerung bereichert wurden. Das Potenzial der Differenzierung der dermalen SSEA-4(+) Zellen, erschien dagegen Estrogenen beschr&auml;nkt werden. Diese Ergebnisse zeigen, dass bestimmte Zelle Bev&ouml;lkerungen mit Variablen potenziellen Mesenchymale Differenzierung von menschlicher Haut isoliert werden k&ouml;nnen. Dar&uuml;ber hinaus haben wir drei verschiedene Teilmengen von dermalen Mesenchymale Progenitorzellen identifiziert.

Menschliche Haut birgt unterschiedliche mesenchymal stromal Zelle Teilmengen.

The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6P-IGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2R-silencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18-36 derived from the Plg-binding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates.

Dissecting mannose 6-phosphate-insulin-like growth factor 2 receptor complexes that control activation and uptake of plasminogen in cells.

The skin has to effectively combat external attacks, while maintaining skin immune homeostasis under steady-state conditions. To fulfill these challenging tasks, the dermis harbors a variety of heterogeneous cell types that are able to suppress T-cell proliferation similar to bone marrow mesenchymal stromal cells. Here we show that plastic-adherent, human dermal cells induce FoxP3 expression in TCR-complex-stimulated CD25(-)CD4(+)CD45RA(+) T cells in the absence of CD28 co-ligation in a cell-contact-dependent manner. These FoxP3(+) T cells reveal an effective suppressive capacity in vitro. Moreover, we found that the vast majority of CD90(+) dermal cells are perivascularly located and generate a significantly higher percentage of regulatory T cells compared with cells expressing markers such as CD271 in vitro. Importantly, we further demonstrate that plastic-adherent dermal cells are also able to differentiate toward the endothelial lineage. Our data show that human skin harbors specific cell types with immunosuppressive potential, which are located in close vicinity to their likely operational area and provide evidence for a CD28-independent regulatory mechanism. Further, the differentiation potential into endothelial cells suggests the existence of a tissue-resident cell pool for vessel regeneration. These findings might have important implications for the clinical use of allogeneic dermal cells to rebuild an imbalanced human skin immune homeostasis.

CD90(+) human dermal stromal cells are potent inducers of FoxP3(+) regulatory T cells.

The spatial and temporal organization of T cell signaling molecules is increasingly accepted as a crucial step in controlling T cell activation. CD222, also known as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is the central component of endosomal transport pathways. In this study, we show that CD222 is a key regulator of the early T cell signaling cascade. Knockdown of CD222 hampers the effective progression of TCR-induced signaling and subsequent effector functions, which can be rescued via reconstitution of CD222 expression. We decipher that Lck is retained in the cytosol of CD222-deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphorylation signature on Lck at the steady state. Hence, CD222 specifically controls the balance between active and inactive Lck in resting T cells, which guarantees operative T cell effector functions.

The late endosomal transporter CD222 directs the spatial distribution and activity of Lck.

[MUV researcher of the month, March 2015].

Upon generation of monoclonal antibodies to the T cell antigen receptor/CD3 (TCR/CD3) complex, we isolated mAb MT3, whose reactivity correlates inversely with the production of IFN-γ by human peripheral blood T lymphocytes. Using eukaryotic expression cloning, we identified the MT3 antigen as myelin-and-lymphocyte (MAL) protein. Flow cytometry analysis demonstrates high surface expression of MAL on all naïve CD4 T cells whereas MAL expression is diminished on central memory- and almost lost on effector memory T cells. MAL T cells proliferate strongly in response to stimulation with CD3/CD28 antibodies, corroborating that MAL T cells are naïve and MAL T cells memory subtypes. Further, resting MAL T cells harbor a larger pool of Ser59- and Tyr394- double phosphorylated lymphocyte-specific kinase (Lck), which is rapidly increased upon in vitro restimulation. Previously, lack of MAL was reported to prevent transport of Lck, the key protein tyrosine kinase of TCR/CD3 signaling to the cell membrane, and to result in strongly impaired human T cell activation. Here, we show that knocking out MAL did not significantly affect Lck membrane localization and immune synapse recruitment, or transcriptional T cell activation. Collectively, our results indicate that loss of MAL is associated with activation-induced differentiation of human T cells but not with impaired membrane localization of Lck or TCR signaling capacity.

Differentiation and activation of human CD4 T cells is associated with a gradual loss of myelin and lymphocyte protein.

Research on cell migration and interactions with the extracellular matrix (ECM) was mostly focused on 2D surfaces in the past. Many recent studies have highlighted differences in migratory behaviour of cells on 2D surfaces compared to complex cell migration modes in 3D environments. When embedded in 3D matrices, cells constantly sense the physicochemical, topological and mechanical properties of the ECM and adjust their behaviour accordingly. Changes in the stiffness of the ECM can have effects on cell morphology, differentiation and behaviour and cells can follow stiffness gradients in a process called durotaxis. Here we introduce a detailed protocol for the assembly of 3D matrices consisting of collagen I/fibronectin and embedding cells for live cell imaging. Further, we will show how the matrix can be stiffened via non-enzymatic glycation and how collagen staining with fluorescent dyes allows simultaneous imaging of both matrix and cells. This approach can be used to image cell migration in 3D microenvironments with varying stiffness, define cell-matrix interactions and the cellular response to changing ECM, and visualize matrix deformation by the cells.

Imaging of Human Cancer Cells in 3D Collagen Matrices.

The extracellular matrix (ECM) is an integral component of all organs and plays a pivotal role in tissue homeostasis and repair. While the ECM was long thought to mostly have passive functions by providing physical stability to tissues, detailed characterization of its physical structure and biochemical properties have uncovered an unprecedented broad spectrum of functions. It is now clear that the ECM not only comprises the essential building block of tissues but also actively supports and maintains the dynamic interplay between tissue compartments as well as embedded resident and recruited inflammatory cells in response to pathologic stimuli. On the other hand, certain pathogens such as bacteria and viruses have evolved strategies that exploit ECM structures for infection of cells and tissues, and mutations in ECM proteins can give rise to a variety of genetic conditions. Here, we review the composition, structure and function of the ECM in cutaneous homeostasis, inflammatory skin diseases such as psoriasis and atopic dermatitis as well as infections as a paradigm for understanding its wider role in human health.

The Extracellular Matrix in Skin Inflammation and Infection.

Keloids are disfiguring, hypertrophic scars with yet poorly understood pathomechanisms, which could lead to severe functional impairments. Here we analyzed the characteristics of keloidal cells by single cell sequencing and discovered the presence of an abundant population of Schwann cells that persisted in the hypertrophic scar tissue after wound healing. In contrast to normal skin, keloidal Schwann cells show a unique, pro-fibrotic phenotype. Our data support the hypothesis that keloidal Schwann cells contribute to the formation of the extracellular matrix and are able to affect M2 polarization of macrophages. Indeed, we show that macrophages in keloids predominantly display a M2 polarization and produce factors that inhibit Schwann cell differentiation. This study suggests the contribution of a Schwann cell - macrophage cross-talk to the continuous expansion of keloids, and that targeting Schwann cells might represent an interesting novel treatment option for keloids.

Schwann cells contribute to keloid formation.

Neutrophil extracellular trap (NET)-formation represents an important defence mechanism for the rapid clearance of infections. However, exaggerated NET formation has been shown to negatively affect tissue-regeneration after injury. As our previous studies revealed the strong tissue-protective and regenerative properties of the secretome of stressed peripheral blood mononuclear cells (PBMCsec), we here investigated the influence of PBMCsec on the formation of NETs. The effect of PBMCsec on NET formation was assessed ex vivo in ionomycin stimulated neutrophils derived from healthy donors using flow cytometry, image stream analysis, and quantification of released extracellular DNA. The effect of PBMCsec on molecular mechanisms involved in NET formation, including Ca-flux, protein kinase C activity, reactive oxygen species production, and protein arginine deiminase 4 activity, were analysed. Our results showed that PBMCsec significantly inhibited NET formation. Investigation of the different biological substance classes found in PBMCsec revealed only a partial reduction in NET formation, suggesting a synergistic effect. Mechanistically, PBMCsec treatment did not interfere with calcium signalling and PKC-activation, but exerted anti-oxidant activity, as evidenced by reduced levels of reactive oxygen species and upregulation of heme oxygenase 1 and hypoxia inducible-factor 1 in PBMCsec-treated neutrophils. In addition, PBMCsec strongly inhibited the activation of protein arginine deiminase 4 (PAD4), ultimately leading to the inhibition of NET formation. As therapeutics antagonizing excessive NET formation are not currently available, our study provides a promising novel treatment option for a variety of conditions resulting from exaggerated NET formation.

The Effect of Paracrine Factors Released by Irradiated Peripheral Blood Mononuclear Cells on Neutrophil Extracellular Trap Formation.

Skin is an active immune organ where professional antigen-presenting cells such as epidermal Langerhans cells (LCs) link innate and adaptive immune responses. While Reticulon 1A (RTN1A) was recently identified in LCs and dendritic cells in cutaneous and lymphoid tissues of humans and mice, its function is still unclear. Here, we studied the involvement of this protein in cytoskeletal remodeling and immune responses toward pathogens by stimulation of Toll-like receptors (TLRs) in resident LCs (rLCs) and emigrated LCs (eLCs) in human epidermis ex vivo and in a transgenic THP-1 RTN1A cell line. Hampering RTN1A functionality through an inhibitory antibody induced significant dendrite retraction of rLCs and inhibited their emigration. Similarly, expression of RTN1A in THP-1 cells significantly altered their morphology, enhanced aggregation potential, and inhibited the Ca flux. Differentiated THP-1 RTN1A macrophages exhibited long cell protrusions and a larger cell body size in comparison to wild-type cells. Further, stimulation of epidermal sheets with bacterial lipoproteins (TLR1/2 and TLR2 agonists) and single-stranded RNA (TLR7 agonist) resulted in the formation of substantial clusters of rLCs and a significant decrease of RTN1A expression in eLCs. Together, our data indicate involvement of RTN1A in dendrite dynamics and structural plasticity of primary LCs. Moreover, we discovered a relation between activation of TLRs, clustering of LCs, and downregulation of RTN1A within the epidermis, thus indicating an important role of RTN1A in LC residency and maintaining tissue homeostasis.

Interoperability of RTN1A in dendrite dynamics and immune functions in human Langerhans cells.

Discovery of a previously unreported deletion in the CDKN2A gene in a case of familial melanoma in Austria.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by type 2 cytokine-driven skin inflammation and epithelial barrier dysfunction. The latter is believed to allow the increased penetration of chemicals, toxins, and allergens into the skin. House dust mite allergens, particularly Der p 2, are important triggers in sensitized individuals with AD; the precise actions of these allergens in epithelial biology remain, however, incompletely understood. In this study, we compared the effects of the protein allergen Der p 2 and a mix of non-IgE-reactive Der p 2 peptides on skin cells using patch tests in AD patients and healthy participants. We then analyzed mRNA expression profiles of keratinocytes by single-cell RNA-sequencing. We report that existing barrier deficiencies in the non-lesional skin of AD patients allow deep penetration of Der p 2 and its peptides, leading to local microinflammation. Der p 2 protein specifically upregulated genes involved in the innate immune system, stress, and danger signals in suprabasal KC. Der p 2 peptides further downregulated skin barrier genes, in particular the expression of genes involved in cell-matrix and cell-cell adhesion. Peptides also induced genes involved in hyperproliferation and caused disturbances in keratinocyte differentiation. Furthermore, inflammasome-relevant genes and IL18 were overexpressed, while KRT1 was downregulated. Our data suggest that Der p 2 peptides contribute to AD initiation and exacerbation by augmenting hallmark features of AD, such as skin inflammation, barrier disruption, and hyperplasia of keratinocytes.

Non-IgE-reactive allergen peptides deteriorate the skin barrier in house dust mite-sensitized atopic dermatitis patients.

Psoriasis is a common chronic inflammatory skin disease characterized by a thickened epidermis with elongated rete ridges and massive immune cell infiltration. It is currently unclear what impact mechanoregulatory aspects may have on disease progression. Using multiphoton second harmonic generation microscopy, we found that the extracellular matrix was profoundly reorganized within psoriatic dermis. Collagen fibers were highly aligned and assembled into thick, long collagen bundles, whereas the overall fiber density was reduced. This was particularly pronounced within dermal papillae extending into the epidermis. Furthermore, the extracellular matrix-modifying enzyme lysyl oxidase was highly upregulated in the dermis of patients with psoriasis. In vitro experiments identified a previously unreported link between hypoxia-inducing factor 1 stabilization and lysyl oxidase protein regulation in mechanosensitive skin fibroblasts. Lysyl oxidase secretion and activity directly correlated with substrate stiffness and were independent of hypoxia and IL-17. Finally, single-cell RNA-sequencing analysis identified skin fibroblasts expressing high amounts of lysyl oxidase and confirmed elevated hypoxia-inducing factor 1 expression in psoriasis. Our findings suggest a potential yet undescribed mechanical aspect of psoriasis. Deregulated mechanical forces hence may be involved in initiating or maintaining of a positive feedback loop in fibroblasts and contribute to tissue stiffening and diminished skin elasticity in psoriasis, potentially exacerbating disease pathogenesis.

Article	Year
Human dermis harbors distinct mesenchymal stromal cell subsets. The Journal of investigative dermatology\| PubMed ID: 22048731	2012
Dissecting mannose 6-phosphate-insulin-like growth factor 2 receptor complexes that control activation and uptake of plasminogen in cells. The Journal of biological chemistry\| PubMed ID: 22613725	2012
CD90(+) human dermal stromal cells are potent inducers of FoxP3(+) regulatory T cells. The Journal of investigative dermatology\| PubMed ID: 25050599	2015
The late endosomal transporter CD222 directs the spatial distribution and activity of Lck. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 25127865	2014
[MUV researcher of the month, March 2015]. Wiener klinische Wochenschrift\| PubMed ID: 25794565	2015
Differentiation and activation of human CD4 T cells is associated with a gradual loss of myelin and lymphocyte protein. European journal of immunology\| PubMed ID: 33345332	2021
Imaging of Human Cancer Cells in 3D Collagen Matrices. Bio-protocol\| PubMed ID: 33732778	2021
The Extracellular Matrix in Skin Inflammation and Infection. Frontiers in cell and developmental biology\| PubMed ID: 34295891	2021
Schwann cells contribute to keloid formation. Matrix biology : journal of the International Society for Matrix Biology\| PubMed ID: 35278628	2022
The Effect of Paracrine Factors Released by Irradiated Peripheral Blood Mononuclear Cells on Neutrophil Extracellular Trap Formation. Antioxidants (Basel, Switzerland)\| PubMed ID: 36009277	2022
Interoperability of RTN1A in dendrite dynamics and immune functions in human Langerhans cells. eLife\| PubMed ID: 36223176	2022
Discovery of a previously unreported deletion in the CDKN2A gene in a case of familial melanoma in Austria. European journal of cancer (Oxford, England : 1990)\| PubMed ID: 36628896	2023
Non-IgE-reactive allergen peptides deteriorate the skin barrier in house dust mite-sensitized atopic dermatitis patients. Frontiers in cell and developmental biology\| PubMed ID: 37675143	2023
Stiffness-Dependent Lysyl Oxidase Regulation through Hypoxia-Inducing Factor 1 Drives Extracellular Matrix Modifications in Psoriasis. The Journal of investigative dermatology\| PubMed ID: 39603411	2025
Editorial: Extracellular matrix modifications in development and disease. Frontiers in cell and developmental biology\| PubMed ID: 39981096	2025

Karin Pfisterer

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