organotypic-slice-cultures-as-preclinical-models-tumor

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate ...

University of Lübeck

Fraunhofer Research and Development Center for Marine and Cellular Biotechnology

Vigilin, a member of the KH protein family, is exceptional among these proteins as it contains 14 KH domains in consecutive order. Vigilin is present in the nucleus and the cytoplasm of all eucaryotic cells studied so far and has apparently high affinity to tRNA and mRNA. There is circumstantial evidence that vigilin expression parallels high translational activity as demonstrated for pancreatic cells in vitro and in vivo as well as for carcinoma cell lines. On a molecular level we have recently demonstrated that vigilin promotes in vitro the export of tRNA from the nucleus to the translational machinery in the cytoplasm and may hence function as an intercompartimental conveyor. In the present study we show that exposure to a vigilin antisense oligo DNA (VAOD) expectedly resulted in a decrease of vigilin-expression, and was concomitant to lower amylase- and trypsin synthesis in freshly isolated pancreatic cells. In addition, carcinoma cells reacted with an increased mortality under exposure to VAOD giving further support for the notion that vigilin participates in cellular life-sustaining processes such as protein translation.

Protein synthesis of eucaryotic cells could be decreased by antisense-DNA of the multi KH domain protein vigilin.

Pancreatitis goes along with changes in exocrine enzyme synthesis and secretion in pancreatic acini. The multi-KH domain protein vigilin is supposed to play an important role in t-RNA trafficking especially in cells with high protein synthesis rates and may reflect the degree of stimulation of translational machinery during pathological processes. In relation to these phenomena we explored in this connection the impact of two different inflammation mediators in a system of isolated rat pancreatic acini.

Vigilin and enzyme expression in isolated pancreatic acini after mellitin and gamma-interferon treatment.

The biological relevance of vigilin a ubiquitous multi (KH)-domain protein is still barely understood. Investigations over the last years, however, provided evidence for a possible involvement of vigilin in the nucleo-cytoplasmic transport of tRNA and in the subsequent association of tRNA with ribosomes. We therefore investigated the potential association of vigilin with 80S ribosomes. Immunostaining, gel filtration, westernblot analysis of polyribosomes and high salt treatment of 80S ribosomes isolated from fresh human placenta were applied to analyze the possible association of vigilin with ribosomes. Overlay assays were performed to examine whether vigilin is capable of binding to ribosomal proteins. Immunostaining of HEp-2 cells, gel filtration of a cytoplasmic extract of HEp-2 cells and westernblot analysis of isolated 80S ribosomes clearly demonstrate that vigilin is bond to the ribosomal complex. Vigilin detaches from the ribosomal complex under the influence of high salt concentrations. We present data that radioactively labeled human vigilin interacts directly with a subset of ribosomal proteins from both subunits. We were able to narrow down the putative binding region to the C-terminal domain by using vigilin mutant constructs. Therefore our results provide strong evidence that vigilin is bond to the ribosomal complex and underline the hypothesis that vigilin might be involved in the link between tRNA-export and the channeled tRNA-cycle on ribosomes.

Vigilin is co-localized with 80S ribosomes and binds to the ribosomal complex through its C-terminal domain.

Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue.

Ultrastructural analysis of mouse embryonic stem cell-derived chondrocytes.

The discovery of epithelial stem cells (eSCs) in the bulge region of the outer root sheath of hair follicles in mice and man has encouraged research into utilizing the hair follicle as a therapeutic source of stem cells (SCs) for regenerative medicine, and has called attention to the hair follicle as a highly instructive model system for SC biology. Under physiological circumstances, bulge eSCs serve as cell pool for the cyclic regeneration of the anagen hair bulb, while they can also regenerate the sebaceous gland and the epidermis after injury. More recently, melanocyte SCs, nestin+, mesenchymal and additional, as yet ill-defined "stem cell" populations, have also been identified in or immediately adjacent to the hair follicle epithelium, including in the specialized hair follicle mesenchyme (connective tissue sheath), which is crucial to wound healing. Thus the hair follicle and its adjacent tissue environment contain unipotent, multipotent, and possibly even pluripotent SC populations of different developmental origin. It provides an ideal model system for the study of central issues in SC biology such as plasticity and SC niches, and for the identification of reliable, specific SC markers, which distinguish them from their immediate progeny (e.g. transient amplifying cells). The current review attempts to provide some guidance in this growing maze of hair follicle-associated SCs and their progeny, critically reviews potential or claimed hair follicle SC markers, highlights related differences between murine and human hair follicles, and defines major unanswered questions in this rapidly advancing field.

Hair follicle stem cells: walking the maze.

Cutaneous nestin+ cells are of substantial interest in regenerative medicine. However, the location of nestin+ cells in situ remains controversial. We therefore sought to determine their location in female human scalp skin, using stringently controlled immunohistochemical techniques, Western blot analysis, and in situ hybridization and complementing those techniques with relative and quantitative reverse transcriptase-PCR of enzymatically digested or laser-capture microdissected human hair follicle (HF) compartments. We show here that the immunoreactivity (IR) patterns obtained with anti-nestin antibodies are highly dependent on the tissue-fixation and immunohistochemical methods used. NESTIN mRNA could not be detected within HF-associated epithelial cells in situ or in RNA extracts of the microdissected HF epithelium. Instead, NESTIN transcripts were found only in intramesenchymal skin compartments. Individual cells showing both, specific nestin IR and NESTIN mRNA were detectable in the connective-tissue sheaths of human HFs, sebaceous and sweat glands. Moreover, stimulation of organ-cultured human scalp skin with the adipokine leptin increased the number of nestin+ cells in these intramesenchymal skin locations, whereas no specific nestin IR could be induced by leptin within the HF epithelium, including the bulge. Therefore, nestin expression at the gene and protein levels in human scalp skin is restricted to the periappendage mesenchyme and can be stimulated by leptin.

Nestin in human skin: exclusive expression in intramesenchymal skin compartments and regulation by leptin.

Clinical success in tissue regeneration requires improvements in vascularization capacity of scaffolds. Several efforts have been made in this field including cellular and acellular technologies. In this work we combined the use of stem cells derived from pancreas or submandibular glands expressing green fluorescent protein (GFP(+)) with a commercially available scaffold for dermal regeneration. Cells were isolated, characterized and seeded in a scaffold for dermal regeneration. Scaffolds containing cells were used to induce dermal regeneration in a full skin defect model. After 3 weeks of in vivo regeneration, tissues were harvested and vascularization was analyzed. Results showed that gland-derived stem cells displayed stem cell features and presented multipotential differentiation capacity because they were able to differentiate in cell types representing the 3 different germ layers. After seeding, cells were homogeneously distributed and formed focal adhesions with the scaffold. Metabolic assays showed that cells can be cultured for at least 3 weeks in the scaffold. In vivo, the presence of pancreatic or submandibular stem cells significantly enhanced the vascularization compared to empty scaffolds. Presence of gland-derived stem cells in the regenerating tissue was confirmed by the detection of GFP expression in the wound area. In order to explore the possible mechanisms behind the improvement in vascular regeneration, in vitro experiments were performed, showing that gland-derived stem cells could contribute by angiogenic and vasculogenic mechanisms to this process. Our results suggest that the combined use of stem cells derived from glands and scaffold for dermal regeneration could be a rational alternative to improve vascularization in scaffold-mediated dermal regeneration.

The use of glandular-derived stem cells to improve vascularization in scaffold-mediated dermal regeneration.

Human skin-derived Nestin+ cells serve as a convenient source for autologous, adult, pluripotent progenitor cells that offer new therapeutic possibilities in cell-based regenerative medicine. However, the isolation of human Nestin+ cells has tended to be of very low efficiency and to produce highly variable cell yields. Here we report a standardised protocol that facilitates the isolation and enrichment of Nestin+ progenitor cells from enzymatically digested adult human scalp dermis. The use of distinct media like Dulbecco's modified Eagle medium supplemented with foetal bovine serum or, alternatively, serum-free, supplemented neural stem cell medium greatly affected cell morphology, proliferation and differentiation (e.g. towards a neural versus mesenchymal phenotype). Finally, Nestin+ cells were isolated from a heterogeneous dermis-derived progenitor cell population, which proliferates within clones or floating microspheres under defined serum-free culture conditions. Supplementation of the medium with epidermal growth factor and basic fibroblast growth factor as well as coating with fibronectin allowed the highest enrichment level of Nestin+ progenitors and differentiation towards neural fate. These methodological advances should greatly facilitate the isolation, culture and targeted differentiation of primary, adult human scalp skin dermis-derived Nestin+ cells.

An improved, standardised protocol for the isolation, enrichment and targeted neural differentiation of Nestin+ progenitors from adult human dermis.

Targeted differentiation of stem cells is mainly achieved by the sequential administration of defined growth factors and cytokines, although these approaches are quite artificial, cost-intensive and time-consuming. We now present a simple xenogeneic rat brain co-culture system which supports neuronal differentiation of adult human stem cells under more in vivo-like conditions.

A novel xenogeneic co-culture system to examine neuronal differentiation capability of various adult human stem cells.

Automated microscopy is currently the only method to non-invasively and label-free observe complex multi-cellular processes, such as cell migration, cell cycle, and cell differentiation. Extracting biological information from a time-series of micrographs requires each cell to be recognized and followed through sequential microscopic snapshots. Although recent attempts to automatize this process resulted in ever improving cell detection rates, manual identification of identical cells is still the most reliable technique. However, its tedious and subjective nature prevented tracking from becoming a standardized tool for the investigation of cell cultures. Here, we present a novel method to accomplish automated cell tracking with a reliability comparable to manual tracking. Previously, automated cell tracking could not rival the reliability of manual tracking because, in contrast to the human way of solving this task, none of the algorithms had an independent quality control mechanism; they missed validation. Thus, instead of trying to improve the cell detection or tracking rates, we proceeded from the idea to automatically inspect the tracking results and accept only those of high trustworthiness, while rejecting all other results. This validation algorithm works independently of the quality of cell detection and tracking through a systematic search for tracking errors. It is based only on very general assumptions about the spatiotemporal contiguity of cell paths. While traditional tracking often aims to yield genealogic information about single cells, the natural outcome of a validated cell tracking algorithm turns out to be a set of complete, but often unconnected cell paths, i.e. records of cells from mitosis to mitosis. This is a consequence of the fact that the validation algorithm takes complete paths as the unit of rejection/acceptance. The resulting set of complete paths can be used to automatically extract important biological parameters with high reliability and statistical significance. These include the distribution of life/cycle times and cell areas, as well as of the symmetry of cell divisions and motion analyses. The new algorithm thus allows for the quantification and parameterization of cell culture with unprecedented accuracy. To evaluate our validation algorithm, two large reference data sets were manually created. These data sets comprise more than 320,000 unstained adult pancreatic stem cells from rat, including 2592 mitotic events. The reference data sets specify every cell position and shape, and assign each cell to the correct branch of its genealogic tree. We provide these reference data sets for free use by others as a benchmark for the future improvement of automated tracking methods.

A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters.

Human skin harbours multiple different stem cell populations. In contrast to the relatively well-characterized niches of epidermal and hair follicle stem cells, the localization and niches of stem cells in other human skin compartments are as yet insufficiently investigated. Previously, we had shown in a pilot study that human sweat gland stroma contains Nestin-positive stem cells. Isolated sweat gland stroma-derived stem cells (SGSCs) proliferated in vitro and expressed Nestin in 80% of the cells. In this study, we were able to determine the precise localization of Nestin-positive cells in both eccrine and apocrine sweat glands of human axillary skin. We established a reproducible isolation procedure and characterized the spontaneous, long-lasting multipotent differentiation capacity of SGSCs. Thereby, a pronounced ectodermal differentiation was observed. Moreover, the secretion of prominent cytokines demonstrated the immunological potential of SGSCs. The comparison to human adult epidermal stem cells (EpiSCs) and bone marrow stem cells (BMSCs) revealed differences in protein expression and differentiation capacity. Furthermore, we found a coexpression of the stem cell markers Nestin and Iα6 within SGSCs and human sweat gland stroma. In conclusion the initial results of the pilot study were confirmed, indicating that human sweat glands are a new source of unique stem cells with multilineage differentiation potential, high proliferation capacity and remarkable self renewal. With regard to the easy accessibility of skin tissue biopsies, an autologous application of SGSCs in clinical therapies appears promising. 

Multipotent nestin-positive stem cells reside in the stroma of human eccrine and apocrine sweat glands and can be propagated robustly in vitro.

High numbers of adult stem cells are still required to improve the formation of new vessels in scaffolds to accelerate dermal regeneration. Recent data indicate a benefit for vascularization capacity by stimulating stem cells with lipopolysaccharide (LPS). In this study, stem cells derived from human skin (SDSC) were activated with LPS and seeded in a commercially available dermal substitute to examine vascularization in vivo. Besides, in vitro assays were performed to evaluate angiogenic factor release and tube formation ability. Results showed that LPS-activated SDSC significantly enhanced vascularization of the scaffolds, compared to unstimulated stem cells in vivo. Further, in vitro assays confirmed higher secretion rates of proangiogenic as well as proinflammatoric factors in the presence of LPS-activated SDSC. Our results suggest that combining activated stem cells and a dermal substitute is a promising option to enhance vascularization in scaffold-mediated dermal regeneration. 

LPS-Stimulated Human Skin-Derived Stem Cells Enhance Neo-Vascularization during Dermal Regeneration.

The human skin fulfills important barrier, sensory, and immune functions-all of which contribute significantly to health and organism integrity. Widespread skin damage requires immediate treatment and coverage because massive skin loss fosters the invasion of pathogens, causes critical fluid loss, and may ultimately lead to death. Since the skin is a highly immunocompetent organ, autologous transplants are the only viable approach to permanently close a widespread skin wound. Despite the development of tissue-saving autologous transplantation techniques such as mesh and Meek grafts, treatment options for extensive skin damage remain severely limited. Yet, the skin is also a rich source of stem and progenitor cells. These cells promote wound healing under physiological conditions and are potential sources for tissue engineering approaches aiming to augment transplantable tissue by generating cultured epidermal autografts (CEAs). Here, we review autologous tissue engineering strategies as well as transplantation products based on skin-derived stem cells. We further provide an overview of clinical trial activities in the field and discuss relevant translational and clinical challenges associated with the use of these products.

Skin-Derived Stem Cells for Wound Treatment Using Cultured Epidermal Autografts: Clinical Applications and Challenges.

The interaction of peripheral nerves with different cells of the skin is a relevant aspect of many physiological processes including nociception, temperature control, and wound healing. Here we describe a protocol for the setup of an indirect co-culture system of peripheral nerve cells and sweat gland-derived stem cells, which can be used to quantify neurite outgrowth.

Fabrication of a Co-Culture System with Human Sweat Gland-Derived Cells and Peripheral Nerve Cells.

Sweat glands play an important role in skin physiology and are an integral part of the natural skin barrier. In order to maintain functionality throughout life, sweat glands make use of several types of stem cells. This chapter focuses on the classification of different types of stem cells found in the sweat gland and their physiological roles. First, sweat gland formation during skin maturation is addressed in order to give an overview of sweat gland origin and formation in vivo. Then, different kinds of adult sweat gland stem cells are introduced and classified between different potency levels and corresponding physiological roles. Finally, the importance of these cell sources for future developments, including applications in wound healing and cosmetics research, is discussed.

Article	Year
Protein synthesis of eucaryotic cells could be decreased by antisense-DNA of the multi KH domain protein vigilin. International journal of molecular medicine\| PubMed ID: 12792806	2003
Vigilin and enzyme expression in isolated pancreatic acini after mellitin and gamma-interferon treatment. Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.]\| PubMed ID: 12890997	2003
Vigilin is co-localized with 80S ribosomes and binds to the ribosomal complex through its C-terminal domain. The international journal of biochemistry & cell biology\| PubMed ID: 15109574	2004
Ultrastructural analysis of mouse embryonic stem cell-derived chondrocytes. Anatomy and embryology\| PubMed ID: 16211392	2005
Hair follicle stem cells: walking the maze. European journal of cell biology\| PubMed ID: 17576022	2007
Nestin in human skin: exclusive expression in intramesenchymal skin compartments and regulation by leptin. The Journal of investigative dermatology\| PubMed ID: 19554024	2009
The use of glandular-derived stem cells to improve vascularization in scaffold-mediated dermal regeneration. Biomaterials\| PubMed ID: 19651436	2009
An improved, standardised protocol for the isolation, enrichment and targeted neural differentiation of Nestin+ progenitors from adult human dermis. Experimental dermatology\| PubMed ID: 20100195	2010
A novel xenogeneic co-culture system to examine neuronal differentiation capability of various adult human stem cells. PloS one\| PubMed ID: 21935488	2011
A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters. PloS one\| PubMed ID: 22087288	2011
Multipotent nestin-positive stem cells reside in the stroma of human eccrine and apocrine sweat glands and can be propagated robustly in vitro. PloS one\| PubMed ID: 24205211	2013
LPS-Stimulated Human Skin-Derived Stem Cells Enhance Neo-Vascularization during Dermal Regeneration. PloS one\| PubMed ID: 26565617	2015
Skin-Derived Stem Cells for Wound Treatment Using Cultured Epidermal Autografts: Clinical Applications and Challenges. Stem cells international\| PubMed ID: 29765411	2018
Fabrication of a Co-Culture System with Human Sweat Gland-Derived Cells and Peripheral Nerve Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 31148084	2019
Heterogeneity of Sweat Gland Stem Cells. Advances in experimental medicine and biology\| PubMed ID: 31487018	2019

Charli Kruse

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