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Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing. This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 µm thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling. OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

The use of in vitro systems to investigate the process of corneal wound healing offers the opportunity to reduce animal pain inflicted during in vivo experimentation. This study aimed to establish an easy-to-handle ex vivo organ culture model with porcine corneas for the evaluation and modulation of epithelial wound healing. Cultured free-floating cornea disks with a punch defect were observed by stereomicroscopic photo documentation. We analysed the effects of different cell culture media and investigated the impact of different wound sizes as well as the role of the limbus. Modulation of the wound healing process was carried out with the cytostatic agent Mitomycin C. The wound area calculation revealed that after three days over 90% of the lesion was healed. As analysed with TUNEL and lactate dehydrogenase assay, the culture conditions were cell protecting and preserved the viability of the corneal tissue. Wound healing rates differ dependent on the culture medium used. Mitomycin C hampered wound healing in a concentration-dependent manner. The porcine cornea ex vivo culture ideally mimics the in vivo situation and allows investigations of cellular behaviour in the course of wound healing. The effect of substances can be studied, as we have documented for a mitosis inhibitor. This model might aid in toxicological studies as well as in the evaluation of drug efficacy and could offer a platform for therapeutic approaches based on regenerative medicine.

BACKGROUND/AIMS: The use of skin-derived stem cells and stem cells of other origins in regenerative medicine requires knowledge of stem cell fate after transplantation. In order to achieve non-invasive long-term imaging and tracking of transplanted stem cells in preclinical studies, a non-toxic, efficient labeling technique that does not alter stem cell characteristics must be used. Our aim was to investigate a method for such a long-term cell-compatible cell tracer using nanoparticles. METHODS: Nanotechnology, in particular the use of quantum dots (QDs), offers great advantages for this crucial requirement. In this study, we used nanocrystals coated with a specific target peptide that enables delivery into the cytoplasm of cells, resulting in an intense and stable fluorescent labeling. We analyzed the influence of biocompatible CdSe/ZnS-QDs on epidermal stem cells (EpiSCs) isolated from adult human skin. Thereby we analyzed on QD loading, cell proliferation including QD transfer to descendent daughter cells as well as the influence on the differentiation potential of stem cells after QD labeling. RESULTS: FACS analysis revealed a dose-dependent QD incorporation into the cells. Thereby, a high initial concentration of nanocrystals resulted in a more stable long-term labeling. QD labeled cells showed normal viability and unchanged ability to proliferate. The spread of QDs during cell division was monitored by time lapse microscopy and two modes of QD distribution could be observed. Daughter cells either received an equal amount of QDs after cell division, which led to a homogenously faded fluorescence signal, or there was an uneven transmission of QDs, which led to unchanged labeling of one cell and a complete loss of the fluorescence signal of the other cell. The spontaneous differentiation potential remained unaffected after QD exposure, since skin-derived EpiSCs showed an unchanged protein and gene expression profile. CONCLUSION: In summary, we can conclude that QDs offer a successful, non-invasive and efficient labeling technique for EpiSCs, which makes their in vitro and in vivo use in skin regeneration and wound healing models traceable. Nevertheless, the uneven transmission of QDs should not be disregarded and the extent and frequency should be investigated in further studies.

The combination of an aging population and an increasing prevalence of diseases associated with impaired-wound healing, including obesity, peripheral vascular disease and diabetes, is likely to result in a dramatic increase in the incidence and prevalence of chronic skin wounds. Indeed, systemic reviews are now not only trying to establish both the prevalence and the often under-estimated socio-economic costs of chronic skin wounds, but most importantly are addressing the impact that chronic wounds have on quality of life. Given the clear need for novel approaches to the management of chronic skin ulceration, ideally developed and tested in the human system in a manner that can be rapidly translated into clinical practice, we examined the effects of multipotent primary human nestin + progenitor cells on human wound healing in an ex vivo model. Human sweat gland-derived nestin + cells demonstrated the capacity to significantly promote two key wound healing parameters, i.e., both reepithelialisation and angiogenesis in experimentally wounded, organ-cultured human skin. The current data further support the use of full-thickness human skin wound-healing models ex vivo to pre-clinically test wound healing-promoting candidate agents. Whilst larger studies are required to substantiate a firm “proof-of-concept,” our preliminary studies encourage further efforts to systemically determine the potential of cell-based regenerative medicine strategies in general, and the use of skin appendage-associated human nestin + cells in particular, as novel treatment strategies for chronic skin ulceration.

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.

A long term functional and reliable coupling between neural tissue and implanted microelectrodes is the key issue in acquiring neural electrophysiological signals or therapeutically excite neural tissue. The currently often used rigid micro-electrodes are thought to cause a severe foreign body reaction resulting in a thick glial scar and consequently a poor tissue-electrode coupling in the chronic phase. We hypothesize, that this adverse effect might be remedied by probes compliant to the soft brain tissue, i.e., replacing rigid electrodes by flexible ones. Unfortunately, this flexibility comes at the price of a low stiffness, which makes targeted low trauma implantation very challenging. In this study, we demonstrate an adaptable and simple method to implant extremely flexible microprobes even to deep areas of rat's brain. Implantation of flexible probes is achieved by rod supported stereotactic insertion fostered by a hydrogel (2% agarose in PBS) cushion on the exposed skull. We were thus able to implant very flexible micro-probes in 70 rats as deep as the rodent's subthalamic nucleus. This work describes in detail the procedures and steps needed for minimal invasive, but reliable implantation of flexible probes.

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.

Background: Beside its anti-proliferative, anti-hypertensive and anti-inflammatory effects, heparin has shown the apoptotic effect in lymphoblasts. In the study, it is aimed to show the apoptotic effect of heparin in lymphoblasts with measuring intracellular calcium and using DNA analysis by flow cytometry, in vitro. Methods: Twenty-three newly diagnosed acute lymphoblastic leukemia patients were included in the study. We added 10 and 20 U/ml heparin into the seperated lymphoblast samples and determined the percentages of apoptosis and intracellular Ca++ levels at 0, 1 and 2 hours by flow cytometry, in vitro. Results: The apoptotic effect on the lymphoblasts were established in 10 and 20 U/ml heparin concentrations at 0, 1 and 2 hours (p=0.005). The apoptotic effect of heparin in lymphoblasts was higher at the first hour than those at 0 and 2 hours in 10 and 20 U/ml heparin concentrations (p=0.005). The highest apoptosis was determined in 20 U/ml heparin concentration at the first hour. Statistically significant increase in intracellular Ca++ levels were determined in 10 and 20 U/ml heparin concentrations at 1 and 2 hours (p=0.005). In 10 and 20 U/ml heparin concentrations, intracellular Ca++ levels were significantly higher at the first hour than 0 and 2 hours (p=0.005). The highest intracellular Ca++ concentration was determined in 20 U/ml heparin concentration at the first hour. Conclusion: Heparin induces apoptosis in lymphoblasts and intracellular Ca++ levels of the lymphoblasts synchronously increase with apoptosis. The increase of intracellular Ca++ level supports a concept that the mitochondria plays a role heparin-induced apoptosis in lymphoblasts.

Minimizing the foreign body response is seen as one critical research strategy for implants especially when designed for immune-privileged organs like the brain. The context of this work is to improve deep brain stimulating devices used in a consistently growing spectrum of psychomotor and psychiatric diseases mainly in form of stiff electrodes. Based on the compliance match hypothesis of biocompatibility we present another step forward using flexible implant materials covered with brain cell-mimicking layers. We covered two types of flexible polyimide films with glandular stem cells derived from pancreatic acini. Using real time-PCR and fluorescent immunocytochemistry we analyzed markers representing various cell types of all three germ layers and stemness. The results demonstrate an unchanged differentiation potential of the polyimide fixated cells as measured by mRNA and protein level. Additionally we developed a fibrinous hydrogel coating to protect them against she ar forces upon eventual implantation. By repeating previous analysis and additional metabolism tests for all stages we corroborate the validity of this improvement. Consequently we assume that a stem cell-containing cover may provide a native, fully and actively integrating brain-mimicking interface to the neuropil.

Vascularization is a key process in tissue engineering and regeneration and represents one of the most important issues in the field of regenerative medicine. Thus, several strategies to improve vascularization are currently under clinical evaluation. In the present study, stem cells derived from hu[for full text, please go to the a.m. URL], 49. Jahrestagung der Österreichischen Gesellschaft für Plastische, Ästhetische und Rekonstruktive Chirurgie (ÖGPÄRC), 42. Jahrestagung der Deutschen Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC), 16. Jahrestagung der Vereinigung der Deutschen Ästhetisch-Plastischen Chirurgen (VDÄPC)

We describe a method for fast and easy isolation of cells via trypsin digestion from lar- vae of Atlantic sturgeon Acipenser oxyrinchus oxyrinchus resulting in a stable, well-proliferating cell culture. The culture conditions for these cells were optimized with the aim of supporting the production of high amounts of biomass. To enhance cell growth and cell density, 4 different cultiva- tion temperatures as well as commercially available carp serum (CS) and fetal calf serum (FCS) at different concentrations were tested and evaluated. Cell growth was measured via an impedance- based online cell-monitoring system (xCELLigence). These results showed the best cultivation tem- perature to be at 25°C and a media composition of Dulbecco's modified Eagle's medium (DMEM) supplemented with either 10 or 20% FCS or 5% CS. The cells were stable in the process of long- term cultivation over 33 passages and could be cryo-preserved. Immunocytochemical analysis re- vealed that the cells expressed proteins of different blastodermic layers. Ectodermic glia fibrilliary acid protein, vigilin (mRNA transport protein), and pan cytokeratin were abundant. This fast- growing cell culture provides an important tool for research on Atlantic sturgeon populations.

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.

BACKGROUND: 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. METHODS AND FINDINGS: This system was applied to well-characterized stem cell populations isolated from human skin, parotid gland and pancreas. In addition to general multi-lineage differentiation potential, these cells tend to differentiate spontaneously into neuronal cell types in vitro and are thus ideal candidates for the introduced co-culture system. Consequently, after two days of co-culture up to 12% of the cells showed neuronal morphology and expressed corresponding markers on the mRNA and protein level. Additionally, growth factors with the ability to induce neuronal differentiation in stem cells could be found in the media supernatants of the co-cultures. CONCLUSIONS: The co-culture system described here is suitable for testing neuronal differentiation capability of numerous types of stem cells. Especially in the case of human cells, it may be of clinical relevance for future cell-based therapeutic applications.

Background/Aims: Cellular models are an interesting tool to study human heart diseases. To date, research groups mainly focus on mouse models, but important murine physiology is different from human characteristics. Recently, scientists found that the electrophysiology of fish cardiomyocytes largely resembles that of humans. So far, cardiomyocyte models were generated using differentiation medium, were stimulated electrically or, when contracting spontaneously, only did so over a short time period. We established an in vitro spontaneously, long-term beating heart model generated from rainbow trout, with the potential to be used as a new human heart model system because of its electrophysiology. Methods: Spontaneously contracting 3D cell layers from rainbow trout were generated in vitro and analyzed using PCR and immunochemistry. Further, electrophysiology was measured via intra – and extracellular recordings. Results: Contracting cardiomyogenic aggregates were generated without differentiation medium and were beating autonomously for more than one month. Electrophysiological measurements exhibit that the action potential properties of fish cardiomyocytes in part resemble the characteristics of human cardiomyocytes. The sensitivity of the beating cell aggregates to drugs could also be confirmed. Conclusion: Spontaneously contracting cardiomyogenic cell aggregates from rainbow trout generated in vitro are suitable for human heart research and pharmacology.

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. Journal of Investigative Dermatology (2009) 129, 2711-2720

Multiple tissue niches in the human body are now recognised to harbour stem cells. Here, we have asked how different adult stem cell populations, isolated from two ontogenetically distinct human organs (skin, pancreas), actually are with respect to a panel of standard markers/characteristics. Here we show that an easily accessible adult human tissue such as skin may serve as a convenient source of adult stem cell-like populations that share markers with stem cells derived from an internal, exocrine organ. Surprisingly, both, human pancreas- and skin-derived stem/progenitor cells demonstrate differentiation patterns across lineage boundaries into cell types of ectoderm (e.g. PGP 9.5+ and GFAP+), mesoderm (e.g. α-SMA+) and entoderm (e.g. amylase+ and albumin+). This intriguing differentiation capability warrants systemic follow-up, since it raises the theoretical possibility that an adult human skin-derived progenitor cell population could be envisioned for possible application in cell replacement therapies.

The molecular principles that lead to chronic pancreatitis are incompletely understood. Trypsin(ogen) plays a key role in the development of pancreatitis. Since the production of trypsin(ogen) by acinary pancreatic cells is paralleled by the expression of vigilin we hypothesised that vigilin may be involved in the onset of pancreatitis. Vigilin is a ubiquitous protein and has apparently high affinity to RNA. In the present study experimental pancreatitis was induced in male rats by a single intravenous application of dibutyltin dichloride (DBTC). Sections of rat pancreas were immunostained with an affinity-purified polyclonal antiserum against vigilin or trypsin(ogen). The changes in vigilin and trypsin(ogen) protein expression were determined by immunoblotting and subsequent sequence analysis of the amino acids. Induction of pancreatitis by DBTC caused alterations in the distribution and the amount of both vigilin and trypsin(ogen) as shown by immunohistochemical and immunoblot analysis. Furthermore we could demonstrate that anionic trypsinogen expression is up-regulated in DBTC-induced chronic pancreatitis. The obtained results suggest that vigilin as well as trypsin(ogen) are involved in the pathogenesis of pancreatitis and that the long-time DBTC-induced pancreatitis is a useful model for study of chronic pancreatitis.

Adult pancreatic stem cells (PSCs) are able to differentiate spontaneously in vitro into various somatic cell types. Stem cells isolated from rat pancreas show extensive self-renewal ability and grow in highly viable long-term cultures. Additionally, these cells express typical stem cell markers such as Oct-4, nestin and SSEA-1. Although differentiation potential is slightly decreasing in long-term cultures, it is possible to keep cell lines up to passage 140. Clonal cell lines could be established from different passages and showed similar characteristics. Remarkably, one clonal cell line, generated from passage 75, showed deviant properties during further culture. Clonal cells formed aggregates, which built tissue-like structures in suspension culture. These generated 3D aggregates produced permanently new cells at the outside margin. Released cells had remarkable size, and closer examination by light microscopy analysis revealed oocyte-like morphology. A comparison of the gene expression patterns between primary cultures of passages 8 and 75, the clonal cell line and the produced oocyte-like cells (OLCs) from tissue-like structures demonstrated some differences. Expression of various germ cell markers, such as Vasa, growth differentiation marker 9 and SSEA-1, increased in the clonal cell line, and OLCs showed additionally expression of meiosis-specific markers SCP3 and DMC1. We here present a first pilot study investigating the putative germ line potential of adult PSCs.

Myocardial regeneration with artificially applied cardiomyocytes is emerging as a promising issue of significant scientific and clinical impact. Nevertheless the source of cells for human cardiomyocyte differentiation especially from adult tissue is still unclear. We hypothesized that human pancreatic stem cells may differentiate into cardiomyocyte-like cells and may increase in number when co-cultured with myocardial tissue. Adult stem cells were harvested from pancreatic tissue of patients undergoing operative procedures including the pancreas. The cells were selected, cultured and passaged. To promote self-differentiation into cardiomyocytes, human pancreatic stem cells were co-cultered with biopsies of human myocardium. After co-culture and breeding, cells were phenotyped as well with respect to RNA, protein and cardiomyocyte specificity at the electron-microscopic level. Pancreatic stem cells have already differentiated spontaneously into cardiomyocyte-like cells performing netlike cell clusters with rare but distinct multilocular cellular autonomous contractions with a frequency of about 20 beats per minute. The number of contracting areas however could be enhanced by co-culture with human myocardial biopsies. On RNA and protein levels as well as in electron-microscopy evidence for cardiomyocyte specificity is shown. To the best of our knowledge this is the first report demonstrating the feasibility of generating autonomously contracting cardiomyocyte-like cells from adult human pancreatic stem cells and their enhancement by myocardial co-culture. This procedure might prove to be an alternative source and method for myocardial regenerative medicine.

Vigilin is a ubiquitous multi heterogeneous nuclear ribonucleoprotein (hnRNP) K homologous (KH)-domain protein. Here we demonstrate that purified recombinant human vigilin binds tRNA molecules with high affinity, although with limited specificity. Nuclear microinjection experiments revealed for the first time that the immuno-affinity-purified nuclear vigilin core complex (VCC(N)) as well as recombinant vigilin accelerate tRNA export from the nucleus in human cells. The nuclear tRNA receptor exportin-t is part of the VCC(N). Elongation factor (EF)-1alpha is enriched in VCC(N) and its cytoplasmic counterpart VCC(C), whereas EF-1beta, EF-1gamma and EF-1delta are basically confined to the VCC(C). Our results suggest further that vigilin and exportin-t might interact during tRNA export, provide evidence that the channeled tRNA cycle is already initiated in the nucleus, and illustrate that intracellular tRNA trafficking is associated with discrete changes in the composition of cellular cytoplasmic multi-protein complexes containing tRNA.

Vigilin, a multidomain hn-ribonucleo-K-homologous protein, is part of a ribonucleoprotein complex with cognate tRNA and is found in both the nucleus and the cytoplasm. In an approach to identify genomic regions involved in regulation of the chicken vigilin gene, we carried out transfection studies with a reporter gene in suitable chicken cells. After including a distantly positioned 5′-sequence in the construct, we observed a 10.5-fold increase in luciferase (EC 1.13.12.7) expression compared with basal promoter activity. Accordingly, chromatin analysis of freshly isolated embryonic tendon fibroblasts with high levels of vigilin mRNA expression shows a DNase-I-hypersensitive site (DHS1) localized 2.2 kb upstream of the transcriptional start site. Similarly, phytohaemagglutinin-stimulated lymphocytes with a 4-fold elevated expression of vigilin mRNA compared with resting lymphocytes also exhibited this unique DHS, having switched from that found at 3.3 kb (DHS2) in resting lymphocytes. Furthermore, using gel-retardation experiments with DNA representing either DHS1 or DHS2, a specific interaction with chicken nuclear extracts was seen.

Vigilin, a protein found predominantly in cells and tissues with a high biosynthetic capacity, was isolated in its native form from human HEp-2 cells (A.T.C.C. CCL23) by immunoaffinity chromatography. Vigilin forms part of a novel ribonucleoprotein complex that also contains additional, as yet uncharacterized, proteins. Experimental evidence suggests that the nucleic acids entrapped in this complex are protected from RNase and belong to the tRNA family. Using either a pool of total human RNA or radioactively labelled tRNA (tRNAAsp**) in rebinding experiments, we could show that tRNA is selectively recaptured by the RNA-depleted vigilin-containing complex.