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.

INTRODUCTION:Nestin-expressing cells isolated from different human tissues reveal self-renewal capacity and a multilineage differentiation potential. In particular, adult stem/progenitor cell populations from exocrine glands such as the pancreas, salivary gland and sweat gland are characterized by prominent nestin expression. Interestingly, human mammary gland histological examinations also demonstrated the existence of nestin-positive cells in the ductal compartments. Within the scope of our previous work we wonder whether an isolation of nestin-positive cell populations from human mammary gland biopsies is possible and what characteristics they have in vitro. Cell populations from both sexes were propagated and subjected to a comparison with other gland-derived cell populations.METHODS:Human mammary tissue biopsies were mechanically and enzymatically treated, and the isolated acini structures were observed with time-lapse microscopy to track adherently outgrowing cells. The proliferation potential of the cell population was assessed by performing growth curves. On the gene and protein levels we investigated the expression of stem cell markers as well as markers indicating multilineage differentiation.RESULTS:We succeeded in establishing proliferating cell populations from breast tissue biopsies of both sexes. Our results display several similarities to the glandular stem cell populations from other exocrine glands. Beside their proliferation capacity during in vitro culture, the obtained cell populations are characterized by their prominent nestin expression. The cells share surface proteins commonly expressed on adult stem cells. We demonstrated the expression of stem cell-related genes like Oct4, Sox2, KLF4 and Nanog, and confirmed multipotent differentiation capacity by detecting transcripts expressed in endodermal, mesodermal and ectodermal cell types.CONCLUSION:With this study we present an efficient procedure for isolation and propagation of nestin-positive stem cells obtained from male and female breast tissue, which is frequently available. The established multipotent cell populations could be easily expanded in vitro and thus hold promise for cell-based therapies and personalized medicine.

With the increasing relevance of cell-based therapies, there is a demand for cell-labeling techniques for in vitro and in vivo studies. For the reasonable tracking of transplanted stem cells in animal models, the usage of quantum dots (QDs) for sensitive cellular imaging has major advances. QDs could be delivered to the cytoplasm of the cells providing intense and stable fluorescence. Although QDs are emerging as favourable nanoparticles for bioimaging, substantial investigations are still required to consider their application for adult stem cells. Therefore, rat pancreatic stem cells (PSCs) were labeled with different concentrations of CdSe quantum dots (Qtracker 605 nanocrystals). The QD labeled PSCs showed normal proliferation and their usual spontaneous differentiation potential in vitro. The labeling of the cell population was concentration dependent, with increasing cell load from 5 nM QDs to 20 nM QDs. With time-lapse microscopy, we observed that the transmission of the QD particles during cell divisions was random, appearing as equal or unequal transmission to daughter cells. We report here that QDs offered an efficient and nontoxic way to label pancreatic stem cells without genetic modifications. In summary, QD nanocrystals are a promising tool for stem cell labeling and facilitate tracking of transplanted cells in animal models.

The current quest for brain-machine-interfaces is hindered by the need to find a reliable solution for truly chronic brain implants. One approach to tackle that problem is not only to use flexible, mechanically compliant micro recording probes, but to enhance their acceptance in the target tissue by biologization. In order to shed light on the best modifications, immunohistochemical staining of long term implants in rat brains provided thousands of images over a six month experimental phase. The current work explains the image processing method, how these images were analysed in the least ambiguous way to provide meaningful and statistically relevant results. Quantitative analysis of the images allowed for the comparison of changes within the image and comparison between different images.

BACKGROUND:Pancreatic cancer is one of the most deadly malignancies with insufficient therapeutic options and poor outcome. Cancer stem cells (CSCs) are thought to be responsible for progression and therapy resistance. We investigated the potential of pancreatic cell lines for CSC research by analyzing to what extent they contain CSC populations and how representative these are compared to clinical tissue.METHODS:Six pancreatic cancer cell lines were analyzed by flow cytometry for CD326, CD133, CD44, CD24, CXCR4 and ABCG2. Subsequently, 70 primary pancreatic tissues were evaluated for CD326, CD133 and CD44 by immunohistochemistry.RESULTS:All the cell lines but one showed a stable expression pattern throughout biological replicates. Marker expression in clinical tissue of CD44 distinguished normal patients from pancreatic carcinoma patients with a sensitivity of 50% at 80% specificity and metastasized from nonmetastasized carcinomas with 69% sensitivity at 100% specificity.CONCLUSIONS:Our results indicate a link between elevated CD44 expression, malignancy and metastasis of pancreatic tissue. Furthermore, individual pancreatic cell lines show a substantial amount of cells with CSC properties which is comparable with interpatient variability detected in primary tissue. These pancreatic cancer cell lines could thus serve for urgently needed pharmacological CSC in vitro research.

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 this study, stem cells derived from human sweat glands were isolated, characterized, seeded in collagen scaffolds, and engrafted in a mouse full skin defect model for dermal regeneration. Results showed that these cells exhibit high proliferation rates and express stem cell and differentiation markers. Moreover, cells responded to angiogenic environments by increasing their migration (P<0.001) and proliferation (P<0.05) capacity and forming capillary-like structures. After seeding in the scaffolds, cells distributed homogeneously, interacting directly with the scaffold, and released bioactive molecules involved in angiogenesis, immune response, and tissue remodeling. In vivo, scaffolds containing cells were used to induce dermal regeneration. Here we have found that the presence of the cells significantly improved vascularization (P<0.001). As autologous sweat gland-derived stem cells are easy to obtain, exhibit a good proliferation capacity, and improve vascularization during dermal regeneration, we suggest that the combined use of sweat gland-derived stem cells and scaffolds for dermal regeneration might improve dermal regeneration in future clinical settings.

Understanding the mechanisms that lead to the differentiation of male germ cells from their spermatogonial stem cells through meiosis to give rise to mature haploid spermatozoa has been a major quest for many decades. Unlike most other cell types this differentiation process is more or less completely dependent upon the cells being located within the strongly structured niche provided by mature Sertoli cells within an intact seminiferous epithelium. While much new information is currently being obtained through the application and description of relevant gene mutations, there is still a considerable need for in vitro models with which to explore the mechanisms involved. Not only are systems of in vitro spermatogenesis important for understanding the basic science, they have marked pragmatic value in offering ex vivo systems for the artificial maturation of immature germ cells from male infertility patients, as well as providing opportunities for the transgenic manipulation of male germ cells. In this review, we have summarized literature relating to simplistic culturing of germ cells, co-cultures of germ cells with other cell types, especially with Sertoli cells, cultures of seminiferous tubule fragments, and briefly mention the opportunities of xenografting larger testicular pieces. The majority of methods are successful in allowing the differentiation of small steps in the progress of spermatogonia to spermatozoa; few tolerate the chromosomal reduction division through meiosis, and even fewer seem able to complete the complex morphogenesis which results in freely swimming spermatozoa. However, recent progress with complex culture environments, such as 3-d matrices, suggest that possibly success is now not too far away.

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.