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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.

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.

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.

Background: We have recently shown that the expression of nestin, a progenitor/stem cell marker protein, is localized in different mesenchymal compartments in human skin including the sweat gland stroma., Objectives: As other exocrine glands are recognized sources of multipotent stem cell populations with potential for multilineage differentiation, it was our aim to isolate, expand and characterize glandular stem cells from human sweat glands., Methods: Isolation of human sweat glands was based on mechanical and enzymatic digestion of axillary skin. Cultivation was performed on collagen-coated cell culture dishes and the resulting cell population was investigated at the protein and mRNA level., Results: Outgrowing cells of isolated sweat glands showed a high-proliferation activity and were characterized by nestin expression in more than 80% of the cells. These sweat gland stem cells could be maintained in culture for long periods of time and showed spontaneous differentiation into cells representative of the different germ layers., Conclusions: This pilot study provides the first, simple protocol for the isolation of adult human nestin-positive stem cells from the sweat gland mesenchyme, which promises to provide an easily accessible and abundantly available, autologous source of multipotent stem cells for cell-based regenerative medicine applications.

Adult stem cells may possess great plasticity, but the cellular mechanisms regulating their fate are not fully understood. Prior to application of stem cell populations in regenerative medicine, major challenges remain to be overcome. Fundamental questions about in vitro growth and spontaneous differentiation of adult stem cell populations must be resolved. In this study, we comprehensively characterized a stem cell population derived from human pancreatic tissue by analyzing mRNA and protein expression in consecutive passages. We examined transcription and protein expression levels of markers related to stem cells or differentiated cells, respectively, as well as the growth rate of a primary human pancreatic stem cell population. In particular, the course of spontaneous mRNA and protein expression of the genes for alpha-smooth muscle actin (alpha-SMA), neurofilaments (NF), cytokeratin 18 (CK18) and nestin was examined during 11 passages by means of RT-PCR and immunocytochemistry. The cell population showed exponential growth over 10 of the 11 examined passages. Both the spontaneous expression of stem cell-related mRNA and protein as well as the characteristics of spontaneous differentiation were variable. Changes in mRNA and protein expression showed no direct correlation. These results demonstrate the unpredictable behaviour of spontaneously differentiating stem cells, being influenced by numerous, barely traceable extrinsic factors. Characterization studies of stem cell populations therefore require improved analysis techniques together with strictly controlled cell cultivation conditions to improve global gene and protein expression analyses.

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.

Cells isolated from pancreas have a remarkable potential for self-renewal and multilineage differentiation. We here present a comprehensive characterisation of stem/progenitor cells derived from exocrine parts of the adult rat pancreas. Using purified cells from either single colonies or even single-cell clones, we specifically demonstrate: (i) the cells contain the typical stem/progenitor cell markers alkaline phophatase, SSEA-1, Oct-4, CD9, Nestin, Pax6, CD44, a-Fetoprotein and Brachyury, demonstrated by immunocytochemistry and RT-PCR; (ii) the cells have the potential to differentiate into lineages of all three germ layers in vitro; (iii) a clonal analysis revealed that even cell lines derived from a single cell have stem/progenitor cell properties such as self-renewal and spontaneous differentiation into various cell lineages; (iv) the cells have the propensity to form three-dimensional, teratoma-like structures in vitro, which contain cells of different lineages; and (v) external stimuli can activate the generation of certain cell types. For instance, cells treated with retinoic acid show an increased expression of alpha-smooth muscle actin. These results suggest that exocrine glands, such as pancreas may be a potential source of adult stem/progenitor cells, suitable for cell therapy of degenerative diseases.

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.