A bioinformatic assay for pluripotency in human cells.

Pluripotent stem cells (PSCs) are defined by their potential to generate all cell types of an organism. The standard assay for pluripotency of mouse PSCs is cell transmission through the germline, but for human PSCs researchers depend on indirect methods such as differentiation into teratomas in immunodeficient mice. Here we report PluriTest, a robust open-access bioinformatic assay of pluripotency in human cells based on their gene expression profiles.

Seminiferous tubule transfection in vitro to define post-meiotic gene regulation.

BACKGROUND: Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. METHODS: Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48 h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment. RESULTS: Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals. CONCLUSION: We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.

Skin-derived human adult stem cells surprisingly share many features with human pancreatic stem cells.

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. alpha-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.

Post-meiotic gene products as targets for male contraception.

Post-meiotic stages of male germ cell maturation represent an interesting target system for the development of novel male contraceptive agents. In the human, these stages represent a period of only about 16 days differentiation, and thus targeting these cells would represent a contraceptive approach with a relatively rapid onset and equivalent recovery. Results from the Human Genome Project suggest that these cells also express a high number of very specific transcripts, though whether all of these are functional and/or essential for sperm differentiation and function requires more research. Until recently, however, these haploid stages were relatively inaccessible to molecular research because of the lack of appropriate model systems and methods. This situation has recently improved, with several new techniques involving manipulation of primary cells and seminiferous tubules, germ cell transplantation and the development of new immortalized cell-lines. Also, new biochemical approaches are yielding more information about haploid-specific transcription factors, such as GCNF. It is therefore to be expected that soon several new targets for a potential post-meiotic male contraceptive will become available for pharmaceutical development.

Mammary gland-derived nestin-positive cell populations can be isolated from human male and female donors.

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.

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

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.

A simple implantation method for flexible, multisite microelectrodes into rat brains.

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.

Models of in vitro spermatogenesis.

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.

The use of human sweat gland-derived stem cells for enhancing vascularization during dermal regeneration.

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.

Cellular modulation of polymeric device surfaces: promise of adult stem cells for neuro-prosthetics.

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 shear 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.

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

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.

The role of alpha-smooth muscle actin in myogenic differentiation of human glandular stem cells and their potential for smooth muscle cell replacement therapies.

IMPORTANCE OF THE FIELD: Cellular replacement therapies in vascular and urogenital organ disorders require an abundant source of smooth muscle cells. A promising approach would be the directed myogenic differentiation (characterized by the expression of alpha-smooth muscle actin (alpha-SMA)) into a sufficient amount of smooth muscle cells through easily obtainable adult stem cells, for example from the sweat gland. AREAS COVERED IN THIS REVIEW: We present novel multipotent adult stem cell populations derived from glandular tissues like pancreas, salivary gland and sweat gland and assess their myogenic potential. Their possible application in cell replacement therapies is discussed, with regard to numerous scaffold-based approaches in the course of the last decade. WHAT THE READER WILL GAIN: Multipotent glandular stem cells can be manipulated by different means to express a predominant smooth muscle-like phenotype. Possible promising applications of myogenic differentiated stem cells were evaluated, since several studies revealed the beneficial effect of somatic stem cells in replacement therapies for blood vessels, bladder reconstructions, etc. TAKE HOME MESSAGE: Glandular stem cells, especially sweat-gland-derived cells, provide an easily accessible and efficient source for autologous smooth muscle tissue, which might be used to replace vascular tissue in case of organ failure or disorder.

Glandular stem cells are a promising source for much more than beta-cell replacement.

Glandular stem cells (GSCs) can be obtained from exocrine glands such as pancreas or salivary glands using well-established cell culturing methods. The resulting cell populations are characterized by a high proliferative capacity and an unusually high plasticity. Cells from pancreas have been demonstrated to differentiate into a multitude of cell types and even into oocyte-like cells. It has been found that the preparation method for GSCs can be applied to many vertebrates, including fishes and birds. Since the cells are excellently cryopreservable, this finding has been utilized to establish a new stem cell bank for preserving living cells of rare and wild animals. Apart from these advances, this mini-review also points out that GSCs from pancreas must not be confused with beta-cell progenitors but constitute a distinct cell type.

Controlling alpha-SMA expression in adult human pancreatic stem cells by soluble factors.

In the application of adult stem cells in regenerative medicine, it is indispensable to control stem cell behaviour in vitro. Since stem cells spontaneously differentiate into several cell types, it is mandatory to identify methods to enrich the desired cell types and concurrently block other differentiation pathways. More precisely, generation of a defined cell population is a key prerequisite for a therapeutic application of stem cells. Here we have demonstrated that it is possible to influence the differentiation of human pancreatic stem cells (hPSCs). During activation of mesodermal differentiation, the cytoskeletal protein alpha-smooth muscle actin (alpha-SMA) seems to play an important role in different cell systems and can usually be detected in hPSCs during in vitro cultivation. We cultured stem cells under different conditions and analyzed the impact on alpha-SMA expression. On the one hand, supplements like retinoic acid (RA) and dimethyl sulfoxide (DMSO) were added to the cultivation medium; on the other hand, different media with or without the addition of fetal calf serum (FCS) were used. Expression of alpha-SMA was determined by immunocytochemistry, Western blot analysis and quantitative RT-PCR. After the treatment of hPSCs with RA, a strong induction of alpha-SMA protein expression was observed when 2mM RA was added to the medium. DMSO in turn induced a marked reduction in alpha-SMA-positive cells. This could also be observed using a keratinocyte serum-free medium (KSFM). Furthermore, the general addition of FCS to the medium had a blocking effect on alpha-SMA expression and decreased the number of alpha-SMA-positive cells to a minimum. The controlled modulation of hPSCs by soluble factors is a first success on the way to a promising application for transplantation medicine and cell therapy of degenerative diseases.

Glandular tissue from human pancreas and salivary gland yields similar stem cell populations.

Stem cells derived from pancreatic tissue are well characterized and exhibit a broad plasticity as they can differentiate beyond lineage boundaries into many cell types. The aim of this study was the comparative characterization of pancreatic stem cells with one other derivate of the embryonic foregut, namely salivary glands, for the existence of similar stem cell populations. The expression of stem cell markers as well as lineage-specific markers was detected by reverse transcription polymerase chain reaction, flow cytometry and immuncytochemical staining. The isolated cells from salivary glands and pancreas grew adherently in vitro and could be maintained for up to 55 and 46 population doublings, respectively. Cells from both tissues showed a comparable phenotype. They expressed different embryonic and adult stem cell markers and had the ability to differentiate spontaneously into cells representing the three embryonic germ layers. Additionally, the directed differentiation of glandular stem cells into the mesodermal lineage was achieved, yielding adipogenic, osteogenic and chondrogenic cells from salivary gland stem cells as well as osteogenic and chondrogenic cells from pancreatic stem cells. Here, we compared two stem cell populations from different glandular tissues which showed similar phenotypes and analogous properties. During embryonic development the two exocrine glands originate from the foregut, which might be the explanation for these intriguing resemblances.

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

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.

Towards the development of a pragmatic technique for isolating and differentiating nestin-positive cells from human scalp skin into neuronal and glial cell populations: generating neurons from human skin?

Nestin+ hair follicle-associated cells of murine skin can be isolated and differentiated in vitro into neuronal and glial cells. Therefore, we have asked whether human skin also contains nestin+ cells, and whether these can be differentiated in vitro into neuronal and/or glial cell populations. In this methodological pilot study, we show that both are indeed the case - employing purposely only very simple techniques for isolating, propagating, and differentiating nestin+ cells from normal human scalp skin and its appendages that do not require selective microdissection and tissue compartment isolation prior to cell culture. We show that, it is in principle, possible to maintain and propagate human skin nestin+ cells for extended passage numbers and to differentiate them into both neuronal (i.e. neurofilament+ and/or PGP9.5+) and glial (i.e. GFAP+, MBP+ and/or O4+) cell populations. Therefore, human scalp skin can serve as a highly accessible, abundant, and convenient source for autologous adult stem cell-like cells that offer themselves to be exploited for neuroregenerative medicine purposes.