Hans-Jürgen Stark's research while affiliated with Universität Heidelberg and other places
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Publications (63)
The BRAF inhibitor vemurafenib, approved for treating patients with BRAF V600E-mutant and unresectable or metastatic melanomas, rapidly induces cutaneous adverse events, including hyperkeratotic skin lesions and cutaneous squamous cell carcinomas (cSCC). To determine, how vemurafenib would provoke these adverse events, we utilized long-term in vitr...
Rocaglates are natural compounds that have been extensively studied for their ability to inhibit translation initiation. Rocaglates represent promising drug candidates for tumor treatment due to their growth-inhibitory effects on neoplastic cells. In contrast to natural rocaglates, synthetic analogues of rocaglates have been less comprehensively ch...
Background: Head and neck squamous cell carcinomas (HNSCC) are phenotypically and molecularly heterogeneous and frequently develop therapy resistance. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and to overcome treatment failure. Methods: We developed a three-dimensional org...
Background. Head and neck squamous cell carcinomas (HNSCC) are histologically and molecularly heterogeneous tumors and frequently develop resistance mechanisms against treatment. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and how to overcome treatment failure.
Methods. We d...
Microsatellite-unstable (MSI) tumours show a high load of mutational neoantigens, as a consequence of DNA mismatch repair deficiency. Consequently, MSI tumours commonly present with dense immune infiltration and develop immune evasion mechanisms. Whether improved lymphocyte recruitment contributes to the pronounced immune infiltration in MSI tumour...
Nonmelanoma skin cancer is the most prevalent cancer worldwide, and a number of cases are steadily increasing. The incidence of skin squamous cell carcinoma, which is the second most frequent tumor in this group of tumors, is markedly increasing in immunosuppressed transplant recipients and under certain cancer treatment modalities. Unfortunately,...
Table S6. 391 Regulated Genes at Day 10 under HIPK4 Knockdown, Related to Figure 3E
Molecular mechanisms responsible for the development of human skin epithelial cells are incompletely understood. As a consequence, the efficiency to establish a pure skin epithelial cell population from human induced pluripotent stem cells (hiPSCs) remains poor. Using an approach including RNAi and high-throughput imaging of early epithelial cells,...
Supplemental material.
Background information on methodology and additional results.
(pdf)
Gene tables.
Spreadsheet file containing the following tables:
Age_MALDR_DE_genes: Listed age-related differential expressed (age MAR) genes.
Age_MALDR_GO: Listed enriched GO terms for age MAR genes.
Age_MALDR_KEGG: Listed enriched KEGG pathways for age MAR genes.
Gender_EQLF_DE_genes: Listed gender related differential expressed (gender DE) genes....
Ageing, the progressive functional decline of virtually all tissues, affects numerous living organisms. Main phenotypic alterations of human skin during the ageing process include reduced skin thickness and elasticity which are related to extracellular matrix proteins. Dermal fibroblasts, the main source of extracellular fibrillar proteins, exhibit...
Age MAR CPM values.
PDF file with summarizeOverlaps derived CPM data for the 42 age-related DE genes.
(pdf)
Age MAR CPM values.
PDF file with gene expression data for the 42 age-related DE genes (derived from edgeR QLF test and ReadExpSet).
(pdf)
Despite decades of skin research, regulation of proliferation and homeostasis in human epidermis is still insufficiently understood. To address the role of mitoses in tissue regulation, we utilized human long-term skin equivalents and systematically assessed mitoses during early epidermal development and long-term epidermal regeneration. We now dem...
Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire genera...
Skin is a complex organ consisting of two very different tissue compartments, the epidermis and the dermis, which comprise cells of diverse origin and function. Establishing and maintaining the functionality of skin requires not only tightly regulated processes of maturation and differentiation of the individual cellular components but also extensi...
Aberrant Wnt regulation, detectable by nuclear translocation of Beta-catenin, is a hallmark of many cancers including skin squamous cell carcinomas (SCCs). By analyzing primary human skin SCCs, we demonstrate that nuclear Beta-catenin is not restricted to SCC cells but also detected in stromal fibroblasts, suggesting an important role for aberrant...
The present invention relates to organotypic cultures of epidermal cells and the use thereof for the screening of pharmaceutical and cosmetic agents. Specifically, means for the improvement of the long-term stability of such cultures are disclosed. Thus, the present invention contemplates a skin equivalent comprising (a) a dermal equivalent compris...
Abbreviations: CP, committed progenitor; D, differentiating cell; K, keratin; KSC, keratinocyte stem cell; LRC, label-retaining cell; TA, transient amplifying; WNT3A, activating Wnt signal; WIF1, Wnt inhibitory factor-1
Understanding intrathymic T cell differentiation has been greatly aided by the development of various reductionist in vitro models that mimic certain steps/microenvironments of this complex process. Most models focused on the faithful in vitro restoration of T cell differentiation and selection. In contrast, suitable in vitro models emulating the d...
The mechanism by which transforming growth factor-β (TGFβ) regulates differentiation in human epidermal keratinocytes is still poorly understood. To assess the role of Smad signaling, we engineered human HaCaT keratinocytes either expressing small interfering RNA against Smads2, 3, and 4 or overexpressing Smad7 and verified impaired Smad signaling...
Telomerase- and telomere length regulation in normal human tissues is still poorly understood. We show here that telomerase is expressed in the epidermis in situ independent of age but was repressed upon the passaging of keratinocytes in monolayer culture. However, when keratinocytes were grown in organotypic cultures (OTCs), telomerase was re-esta...
12R-lipoxygenase (12R-LOX) represents a key enzyme of a recently identified eicosanoid pathway in the skin that plays an essential role in the establishment and/or maintenance of the epidermal barrier function. Genetic studies show that loss-of-function mutations in ALOX12B, encoding 12R-LOX, and in ALOXE3, encoding another closely related LOX invo...
Stem cells in human interfollicular epidermis are still difficult to identify, mainly because of a lack of definitive markers and the inability to label human beings for label-retaining cells (LRCs). Here, we report that LRCs could be identified and localized in organotypic cultures (OTCs) made with human cells. Labeling cultures for 2 weeks with i...
We reported recently the induction of selective iodide uptake in prostate cancer cells (LNCaP) by prostate-specific antigen (PSA) promoter-directed sodium iodide symporter (NIS) expression that allowed a significant therapeutic effect of (131)I. In the current study, we studied the potential of the high-energy alpha-emitter (211)At, also transporte...
Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine pep...
In vitro generated skin models find growing interest as promising tools in basic research and clinical application in regenerative medicine. Here, we present further details of an improved long-term skin equivalent (SE) enabling mechanistic studies on skin reconstruction and epidermal function. Growth conditions of fibroblasts in a 3D scaffold were...
We reported recently the induction of iodide accumulation in prostate cancer cells (LNCaP) by prostate-specific antigen promoter-directed sodium iodide symporter (NIS) expression that allowed a significant therapeutic effect of (131)iodine ((131)I). These data demonstrated the potential of the NIS gene as a novel therapeutic gene, although in some...
Interactions between cancer cells and the tissue microenvironment play an essential role in controlling tumor development and progression. Here, we report that stromal modulation induced by a biodegradable meshwork (Hyalograft 3D) inhibited tumor vascularization and invasion of the locally invasive low-grade malignant human HaCaT-ras II-4 keratinoc...
Excessive basement membrane (BM) deposition in skin and mucosa is characteristic for lipoid proteinosis (LP; hyalinosis cutis et mucosae), an inherited disease caused by extracellular matrix protein 1 (ECM1) mutations. According to ultrastructure there are striking differences between junctional and microvascular BM.
Distinct analysis of the juncti...
Nidogens are considered as classical linkers joining laminin and collagen IV networks in basement membranes (BMs); however, recent genetic approaches have suggested that nidogens function in a tissue-specific and developmental context. Thus, in mice lacking both nidogen-1 and -2 heart and lung were severely affected, causing neonatal death. Further...
Platelet-derived growth factor (PDGF) stimulates tumor growth and progression by affecting tumor and stromal cells. In the HaCaT skin carcinogenesis model, transfection of immortal nontumorigenic and PDGF-receptor-negative HaCaT keratinocytes with PDGF-B induced formation of benign tumors. Here, we present potential mechanisms underlying this tumor...
Epidermal homeostasis is understood as the maintenance of epidermal tissue structure and function by a fine tuned regulatory mechanism balancing proliferation and cell loss by desquamation and apoptosis. The lack of appropriate experimental models has largely prevented a better understanding of the regulatory mechanisms controlling epidermal tissue...
In this chapter, we present technical details for the generation of in vitro skin equivalents consisting of collagen gels with incorporated fibroblasts covered by proliferating and differentiating keratinocytes. Epithelial-mesenchymal interactions are clearly manifest in these skin equivalents. Therefore, they have proven to be suitable experimenta...
Besides medical application as composite skin grafts, in vitro constructed skin equivalents (SEs) or organotypic co-cultures represent valuable tools for cutaneous biology. Major drawbacks of conventional models, employing collagen hydrogels as dermal equivalents (DEs), are a rather poor stability and limited life span, restricting studies to early...
The transcription factors Snail and E47 are direct repressors of E-cadherin, with both inducing a full epithelial-mesenchymal transition and invasive behaviour in vitro when expressed in the prototypic epithelial MDCK cell line. The role of these repressors in the invasive process and in other tumorigenic properties is, nevertheless, still poorly u...
Basement membranes generally determine different tissue compartments in complex organs, such as skin, playing not only an important structural but also a regulatory role. We have previously demonstrated the formation of a regular basement membrane in organotypic three-dimensional (3D)-cocultures of human skin keratinocytes and fibroblasts by indire...
To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, we have established in vitro skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination...
In this chapter, we present technical details for the generation of in vitro skin equivalents consisting of collagen gels with incorporated fibroblasts covered by proliferating and differentiating keratinocytes. Epithelial-mesenchymal interactions are clearly manifest in these skin equivalents. Therefore, they have proven to be suitable experimenta...
Formation of a well structured epidermis strictly depends on a tight balance between proliferation and differentiation. Accordingly, telomerase, which is restricted to proliferating cells, is downregulated with differentiation. It is unclear, however, whether this inhibition is essential to or only a consequence of the differentiation process. By s...
Accumulating evidence indicates a decisive role for the adjacent stroma in tumour growth and dissemination. However, it is not clear how far altered differentiation such as expression of aberrant keratins and vimentin, common in invasive human carcinomas, may reflect intrinsic cell properties or a response to the tumour environment. We have address...
The current study was undertaken to analyse growth and differentiation-related functions of normal keratinocytes (NOK) and an SV40T-immortalized keratinocyte line (SVpgC2a) from buccal mucosa, viewing the latter cell line as a model of a dysplastic epithelium. Morphological and immunohistochemical assessments of organotypic epithelia generated from...
This chapter focuses on the role of dermal fibroblasts in epidermal keratinocyte proliferation and differentiation and proves the usefulness of an organotypic in vitro skin equivalent model to study mechanisms of tissue regeneration. Examples are given how these can be used to study the growth of keratinocytes after transplantation in vivo, alone o...
Balanced keratinocyte proliferation and differentiation resulting in regular tissue organization strictly depend on dermal support. Organotypic cultures represent biologically relevant in vitro models to study the molecular mechanism of the underlying dermal-epidermal interactions. To mimic the state of resting fibroblasts in the dermis, postmitoti...
Skin equivalents formed by keratinocytes cocultured with fibroblasts embedded in collagen lattices represent promising tools for mechanistic studies of skin physiology, for pharmacotoxicologic testing, and for the use as skin substitutes in wound treatment. Such cultures would be superior in defined media to avoid interference with components of se...
The immortal human keratinocyte line HaCaT has been employed in many studies as paradigm for epidermal keratinocytes. In order to demonstrate its potential to form stable epidermal structures in response to connective tissue, this was challenged in surface transplants on nude mice, where normal keratinocytes rebuild a typical epidermis within two w...
The cutaneous basement membrane zone, composed of numerous macromolecules, plays a multifunctional role in tissue regeneration and maintenance. To elucidate the cellular origin and dynamics of basement membrane formation, de novo synthesis, deposition, and ultrastructural assembly of its components were analyzed in organotypic cultures of adult ski...
We studied expression of the 5-lipoxygenase (5-LO) pathway in normal human skin. In situ hybridization revealed a 5-LO mRNA-containing epidermal cell (EC) population that was predominantly located in the midportion of the spinous layer, in outer hair root sheaths, and in the epithelial compartment of sebaceous glands. Examination of skin specimens...
Integrin patterns and formation of basement membrane (BM) were investigated in correlation to epidermal growth and differentiation during skin regeneration in human keratinocyte transplants on nude mice. Immuno-fluorescence and transmission electron microscopy (TEM) showed that different stages of tissue reconstruction were characterized by a seque...
Cell immortalization is supposed to be an early step in the transformation process of human cells. This is usually accompanied by a loss or reduction in the differentiation potential. Nevertheless it has been shown that the spontaneously immortalized human keratinocyte line HaCaT had largely preserved its differentiation capacity and could form a n...
The growth and differentiation response to retinoic acid (RA) was studied in the human keratinocyte line HaCaT and tumorigenic clones transfected with c-Ha-ras oncogene (HaCaT-ras). Differentiation (mainly keratin synthesis) was evaluated and correlated to cell proliferation in vitro but also growth behaviour in vivo (tumorigenicity). Comparable to...
Follicles of human anagen hair were separated into morphologically distinct compartments (by sequential trypsinization and microdissection) for the biochemical and immunological analysis of keratins as differentiation markers to diagnose the type of epithelial differentiation. While outer root sheath contained throughout the "soft" (cyto)keratins K...
In the past, immortalization of human epidermal keratinocytes has been generally considered to have a massive impact on their differentiation properties. This is certainly true for many virally transformed cells. In contrast, the recently described spontaneous cell line HaCaT exhibits a virtually normal growth and differentiation pattern when trans...
In culture, keratinocytes generally express aberrant growth and differentiation programs, which are largely normalized in cell transplants. In order to study the underlying regulatory phenomena and to distinguish between intrinsic properties and external factors, different in vitro and in vivo models have been applied using human keratinocytes from...
Keratins produced by morphologically distinct compartments of the human hair folicle (hHF) were analysed and compared to those produced by cultured hHF and interfollicular keratinocytes. Five of the major keratins, the basic keratins nos. 5 and 6 (apparent mol. mass 60 and 58 kDa) and the acidic keratins nos. 14, 16, and 17 (51, 49 and 48 kDa), cou...
Citations
... Despite numerous attempts, restoring the thymic function has been challenging due to the difficulty to reproduce the thymic microenvironment. Notably, thymic three-dimensional (3-D) configuration is critical to the survival and function of TECs, whereas TECs cultured in a 2-D environment rapidly decrease the expression of genes critical for thymopoiesis 3,4 . ...
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... 23 The activation of the RAS−RAF−MEK−ERK signaling pathway is an important molecular basis for the skin disease and cure. 24 When skin is wounded, the Ras/Raf/ERK/ MEK signaling pathway is also obviously damaged. 25 Skin wound is a strong stimulus, which can be used as a signal to activate the Ras/Raf/MEK/ERK signaling pathway. ...
... The active form of vitamin D, calcitriol (1,25-dihydroxyvitamin D 3 ), decreases both the volume and weight of tumors in rat models by reducing GLUT1, downregulating glycolytic enzymes including HK2 and LDHA, and lowering the expression of cyclin D1 and c-Myc [93]. Recent studies demonstrated that synthetic rocaglate derivatives (IMD-1: IMD-026259, IMD-3: IMD-026260) in combination with glucose anti-metabolite 2-deoxy-D-glucose (2DG) downregulates glucose transporter GLUT-1 expression and induces CRC cell death in vitro via inhibition of glucose metabolism [94]. ...
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... Explants are often used to obtain adherent 2D primary cell cultures, but in the case of the PDE model this is not done, so researchers cultivate explants under special conditions that exclude the migration of plastic-adherent cells from the tissue fragment. To do this, HNSCC explants are placed on an additional supporting matrix, for example, a collagen sponge [20] or a dermal equivalent consisting of human fibroblasts cultured on a viscose fiber fabric [130]. ...
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... (CIN), microsatellite instability (MSI) and CpG island methylation phenotype (CIMP). [3][4][5] CIN is the most classical and common molecular pathway involved in CRC, accounting for 75% of all CRC cases. It is characterised by chromosomal alterations, including somatic copy number alterations due to deletion, aneuploid loss, insertion and amplification. ...
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... Immunosuppression is also a very relevant risk factor for squamous cell carcinoma (SCC), as immunosuppressed transplant recipients bear a 100-fold risk for the development of cutaneous SCC. In these cases, the tumors occur earlier in life and are more aggressive, with a metastasis rate of up to 10% [37]. Cutaneous human papillomavirus of the genus beta is a particular risk factor in SCC. ...
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... In addition, GDF11 is also associated with the inactivation of inflammatory responses and protection against them by inhibiting oxidative stress and the expression of heat shock proteins [47]. Downregulation of homeodomain interaction protein kinase 4 was determined in our analysis; its silencing was found to be related to skin epithelial differentiation from stem cells in a previous study [48]. ...
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... The highest collagen content was found in males older than 40, whereas for women in a 65-year-old donor and some subjects younger than 30 years old, which contradicts the notion of decreased collagen expression in in-vivo aged skin. Kaisers et al. also showed that the changes in gene expression of in-vivo aged skin are not represented in short-term cultures [55]. Furthermore, in the unpublished study mentioned above, we have also seen a 75% increase in COL1A1 gene expression in the older age group compared to the younger group. ...
... Studies on keratinocytes have been greatly facilitated by the fact that these cells can be isolated from mammalian skin and can be easily grown in culture, where they retain much of their in vivo properties [1]. Keratinocytes in culture exhibit polarity, grow as epithelial sheets and can be induced to undergo terminal squamous differentiation [2,3,4]. Therefore, these cells have been widely utilized to study various biological processes of epithelial cells such as cell cycle progression, differentiation, migration, and wound healing [4,5]. ...
... Two experimental strategies enable the study of the in vitro formation of the thymus's 3D structure that reproduces its microenvironment with the extracellular matrix and thymic epithelial cells. One of these strategies is the thymus re-aggregation [19,20], and the other is the 3D organotypic culture [23]. In the re-aggregation model, the thymus tissue is devoid of cells (decellularized), but retains most of the microenvironment of the extracellular matrix that could support the re-aggregation between TECs. ...
