FG Zellbiologie
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The standard screening test for the recognition of autoimmune diseases is the proof of autoantibodies in serum of patients by indirect immunofluorescence (IIF) based on HEp‐2 cells. Manual evaluation of this test is very subjective, slow, and there are no objective parameters as guidelines available. Interlaboratory tests showed occasionally large deviations in the test evaluation resulting in a high variance of results. The aim of this project is fast, objective, safe, and economical automatic analysis of HEp‐2 IIF patterns. Images of IIF patterns were completely and automatically captured using an inverse motorized fluorescence microscope. Thereby, device‐specific parameters were controlled automatically, too. For fast analysis of IIF patterns new algorithms of image processing were developed. Artifacts were recognized and excluded from analysis by the developed software. Analysis of more than 80,000 images clearly demonstrated full automatization and fast processing of IIF patterns. Additionally serum‐specific fluorescence could be easily distinguished from background. Even very weak but positive patterns can be recognized and used for diagnosis. A detailed separation into different basic patterns is possible. Objective, fast, and disease‐related economical analysis of HEp‐2 immunofluorescence patterns is feasible. The implemented software algorithms allowed a mathematical way of describing IIF patterns and can therefore be a useful tool for the needed standardization process.
Trotz Einführung von Multiplex-Assays ist die Bestimmung von Autoantikörpern (AAK) mittels indirekter Immunfluoreszenz (IIF) nach wie vor ein wesentlicher Bestandteil der Autoimmundiagnostik. Das Screening auf nichtorganspezifische AAK wird in der Routinediagnostik von autoimmunen rheumatischen Erkrankungen mittels indirekter Immunfluoreszenz an HEp-2-Zellen durchgeführt. Leider differieren die mit diesem Test erhaltenen Befunde z. T. recht erheblich auf Grund von objektiven (z. B. HEp-2-Zellfixierung) und subjektiven (z. B. Erfahrungswerte) Faktoren. Die Intra- und Interlaborvarianzen sind daher relativ hoch. Erste Ergebnisse von Evaluierungsstudien eines neu entwickelten Systems zur automatischen Bildanalyse (AKLIDES®) zeigen einen Weg in Richtung Standardisierung (bzw. Harmonisierung) des HEp-2-Zell-Assays auf, in dem zunächst subjektive Fehlinterpretationen vermieden werden können. Eine weitere Harmonisierung erfordert die Weiterentwicklung der Software-Algorithmen zur Mustererkennung sowie neue Kalibrationssysteme.
AbstractBackgroundIdentification of antinuclear antibodies (ANAs) has large clinical importance for the assessment of autoimmune diseases. HEp‐2 cell preparations on microscopic slides are commonly used as antigenic substrate. Methods used for cell preparation are important for ANA pattern analysis; however, these methods differ widely and are mostly not specified.MethodsHEp‐2 cells were fixed using acetic acid‐ethanol, methanol‐acetone, acetone, formaldehyde, paraformaldehyde, or glutaraldehyde. Morphological analysis was done after haematoxylin‐eosin staining and DAPI‐staining of cell nuclei.ResultsThe results demonstrate a high variability of cell and nuclear morphology depending on the used fixatives. Aldehyde fixatives conserved the cell structures best, acetone fixatives revealed remarkable changes.ConclusionsAfter selecting appropriate fixation procedures to preserve nuclear structures further experiments are necessary to find out which fixation procedure preserves the disease‐linked antigens the best way and are, therefore, suitable to be used in ANA‐testing of AABs. © 2006 International Society for Analytical Cytology
A dysfunctional miR-1-TRPS1-MYOG axis drives ERMS by suppressing terminal myogenic differentiation
(2023)
Rhabdomyosarcoma is the most common pediatric soft tissue tumor, comprising two major subtypes: the PAX3/7-FOXO1 fusion-negative embryonal and the PAX3/7-FOXO1 fusion-positive alveolar subtype. Here, we demonstrate that the expression levels of the transcriptional repressor TRPS1 are specifically enhanced in the embryonal subtype, resulting in impaired terminal myogenic differentiation and tumor growth. During normal myogenesis, expression levels of TRPS1 have to decrease to allow myogenic progression, as demonstrated by overexpression of TRPS1 in myoblasts impairing myotube formation. Consequentially, myogenic differentiation in embryonal rhabdomyosarcoma in vitro as well as in vivo can be achieved by reducing TRPS1 levels. Furthermore, we show that TRPS1 levels in RD cells, the bona fide model cell line for embryonal rhabdomyosarcoma, are regulated by miR-1 and that TRPS1 and MYOD1 share common genomic binding sites. The myogenin (MYOG) promoter is one of the critical targets of TRPS1 and MYOD1; we demonstrate that TRPS1 restricts MYOG expression and thereby inhibits terminal myogenic differentiation. Therefore, reduction of TRPS1 levels in embryonal rhabdomyosarcoma might be a therapeutic approach to drive embryonal rhabdomyosarcoma cells into myogenic differentiation, thereby generating postmitotic myotubes.
Human serum or FCS: Differentiation of human cartilage-like microtissues depends on serum selection
(2018)
Introduction: Regeneration of articular cartilage defects is limited. Hence, cell-based therapies for the treatment were developed using e.g. chondrocytes isolated from patient biopsies. For cell culture fetal calf serum (FCS) is often used as media supplement to establish engineering processes for transplants in regenerative medicine. However, the usage of xenogeneic serum could mask the intrinsic behavior of human cells in vitro. This could be particularly important for the dedifferentiation process during cell expansion and redifferentiation in 3D-culture. The aim of this study was to compare the development of microtissues derived from chondrocytes cultivated in presence of FCS with those cultivated in human serum (HS).
Material and Methods: Human articular chondrocytes isolated from knee joints were expanded in medium with HS or FCS. Scaffold-free microtissues were generated using the agar overlay technique. After four and eight weeks the macroscopic appearance (reflected-light microscopy) was documented and the size of the microtissues was determined. The differentiation degree was evaluated via histology to visualize typical glycosamino¬glycans (Safranin O, Alcian blue) and immunohisto¬chemistry (IH) to detect cartilage-specific markers (collagen type II, proteoglycans, COMP, Sox9) on cryosections.
Results and Conclusion: Cells from all donors in both medium compositions generated microtissues – but with distinct differences in the differentiation levels. Microtissues cultured in medium with FCS showed a higher differentiation level compared to microtissues in HS. This is evidenced by a higher amount of matrix resulting in bigger microtissues (diameter in FCS is 42% larger compared to HS) and a more cartilage-like composition of the matrix (not any/little colouring in HS versus intense staining in FCS). IH analysis showed the same pattern. Not any/less positivity for collagen type II, proteoglycan, COMP, and Sox9 in HS versus moderate-to-high positivity in FCS-cultured microtissues. Absolute values vary from donor to donor whereas an individualized expression profile only became visible in microtissues cultured in HS medium. HS should be preferred for the engineering of cartilage-like microtissues, as it rather enables a "human-based" situation in vitro.
Supported by „Gesundheitscampus Brandenburg“ and „Ministerium für Wissenschaft, Forschung und Kultur“, state of Brandenburg, Germany.
Differentiation of human chondrocytes under the influence of microalgae-based exopolysaccharides
(2019)
BACKGROUND: Exopolysaccharides are formed by microalgae in response to adverse conditions and are found mainly as a coating around the cells. The protective function of EPS plays an important role in the survival of microalgae in polluted habitats. So far, the particular biological and physico-chemical properties of EPS have not been adequately investigated and their effect on cells such as human chondrocytes has not been tested. Microalgae in particular offer an ideal platform for the production of dietary supplements or drugs that enable new therapeutic approaches.
METHODS: To investigate the influence of EPS on human chondrocytes, cytotoxicity and differentiation potential were analyzed in 3D cultures.
RESULTS: We were able to demonstrate a positive, but donor-specific influence of EPS on the differentiation of human chondrocytes. Furthermore, the EPS showed no cytotoxic effects in a range of up to 250 µg EPS per ml medium.
CONCLUSION: EPS have the potential to positively influence the differentiation of human chondrocytes. Further experiments are necessary to analyze the donor-specific effect.
BACKGROUND: Tissue engineering has become a major field of research in biotechnology and biomedicine. As a consequence, cell-based therapeutic approaches are entering the hospitals, especially for skeletal regeneration. Traumatic injuries of cartilage are treated with autologous cell suspensions or in vitro generated cartilage tissues, but there is actually no therapy available for degenerative cartilage defects. However, Osteoarthritis (OA) is a major public health problem in the world affecting 240 million people globally.
OBJECTIVE: To develop suitable in vitro tissues, the properties of chondrogenic spheroids should be optimized via coculture with cells naturally occurring as joint neighbours.
METHODS: Human chondrocytes were isolated from condyles and propagated in monolayer culture. Scaffold-free spheroids were generated and co-cultured with joint-specific partner cells (osteoblast-like osteosarcoma cells, fibroblasts). Morphology and differentiation was analyzed using histochemistry (Alcian blue, Safranin O) and immunohistochemistry for cartilage markers (collagen type II, Sox9, proteoglycan), proliferation-associated protein (Ki67) and markers of connective tissue (collagen type I and actin).
RESULTS: The provision of a more natural microenvironment in vitro via co-culture of chondrocyte-based aggregates with osteoblast-like Saos-2 cells enhanced the differentiation potential of chondrogenic spheroids towards hyaline cartilage.
CONCLUSIONS: The study showed the positive influence of Saos-2 cells on the differentiation potential of human chondrocytes in co-culture.