Stephan Tiede

Exploring the role of stem cells in cutaneous wound healing

Abstract:  The skin offers a perfect model system for studying the wound healing cascade, which involves a finely tuned interplay between several cell types, pathways and processes. The dysregulation of these factors may lead to wound healing disorders resulting in chronic wounds, as well as abnormal scars such as hypertrophic and keloid scars. As the contribution of stem cells towards tissue regeneration and wound healing is increasingly appreciated, a rising number of stem cell therapies for cutaneous wounds are currently under development, encouraged by emerging preliminary findings in both animal models and human studies. However, we still lack an in‐depth understanding of the underlying mechanisms through which stem cells contribute to cutaneous wound healing. The aim of this review is, therefore, to present a critical synthesis of our current understanding of the role of stem cells in normal cutaneous wound healing. In addition to summarizing wound healing principles and related key molecular and cellular players, we discuss the potential participation of different cutaneous stem cell populations in wound healing, and list corresponding stem cells markers. In summary, this review delineates current strategies, future applications, and limitations of stem cell‐based or stem cell‐targeted therapy in the management of acute and chronic skin wounds.

Immunophenotyping of the human bulge region: the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche

Abstract:  Since the discovery of epithelial hair follicle stem cells (eHFSCs) in the bulge of human hair follicles (HFs) an important quest has started: to define useful markers. In the current study, we contribute to this by critically evaluating corresponding published immunoreactivity (IR) patterns, and by attempting to identify markers for the in situ identification of human eHFSCs and their niche. For this, human scalp skin cryosections of at least five different individuals were examined, employing standard immunohistology as well as increased sensitivity methods. Defined reference areas were compared by quantitative immunohistochemistry for the relative intensity of their specific IR.

Mucolipidosis II is caused by mutations in GNPTA encoding the alpha/beta GlcNAc-1-phosphotransferase

Mucolipidosis II (ML II) is a fatal lysosomal storage disorder resulting from defects in the multimeric GlcNAc-1-phosphotransferase responsible for the initial step in the generation of the mannose 6-phosphate (M6P) recognition marker. M6P residues on oligosaccharides of newly synthesized lysosomal enzymes are essential for efficient receptor-mediated transport to lysosomes. We used the recombinant GlcNAc-1-phosphotransferase γ subunit as an affinity matrix to purify an unknown protein identified as the product of GNPTA (encoding GNPTA, previously known as MGC4170). The cDNA encodes a protein of 1,256 amino acids with two putative transmembrane domains and a complex preserved modular structure comprising at least six domains. The N-terminal domain of GNPTA, interrupted by a long insertion, shows similarities to bacterial capsule biosynthesis proteins. We identified seven mutations in GNPTA that lead to premature translational termination in six individuals with ML II. Retroviral transduction of fibroblasts from an individual with ML II resulted in the expression and localization of GNPTA in the Golgi apparatus, accompanied by the correction of hypersecretion of lysosomal enzymes. Our results provide evidence that GNPTA encodes a subunit of GlcNAc-1-phosphotransferase defective in individuals with ML II.

Mannose phosphorylation in health and disease

Lysosomal hydrolases catalyze the degradation of a variety of macromolecules including proteins, carbohydrates, nucleic acids and lipids. The biogenesis of lysosomes or lysosome-related organelles requires a continuous substitution of soluble acid hydrolases and lysosomal membrane proteins. The targeting of lysosomal hydrolases depends on mannose 6-phosphate residues (M6P) that are recognized by specific receptors mediating their transport to an endosomal/prelysosomal compartment. The key role in the formation of M6P residues plays the GlcNAc-1-phosphotransferase localized in the Golgi apparatus. Two genes have been identified recently encoding the type III alpha/beta-subunit precursor membrane protein and the soluble gamma-subunit of GlcNAc-1-phosphotransferase. Mutations in these genes result in two severe diseases, mucolipidosis type II (MLII) and III (MLIII), biochemically characterized by the missorting of multiple lysosomal hydrolases due to impaired formation of the M6P recognition marker, and general lysosomal dysfunction. This review gives an update on structural properties, localization and functions of the GlcNAc-1-phosphotransferase subunits and improvements of pre- and postnatal diagnosis of ML patients. Further, the generation of recombinant single-chain antibody fragments against M6P residues and of new mouse models of MLII and MLIII will have considerable impact to provide deeper insight into the cell biology of lysosomal dysfunctions and the pathomechanisms underlying these lysosomal disorders.

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?

Abstract:  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.

Thyroid-stimulating hormone, a novel, locally produced modulator of human epidermal functions, is regulated by thyrotropin-releasing hormone and thyroid hormones

Several elements of the hypothalamic-pituitary-thyroid axis (HPT) reportedly are transcribed by human skin cell populations, and human hair follicles express functional receptors for TSH. Therefore, we asked whether the epidermis of normal human skin is yet another extrathyroidal target of TSH and whether epidermis even produces TSH. If so, we wanted to clarify whether intraepidermal TSH expression is regulated by TRH and/or thyroid hormones and whether TSH alters selected functions of normal human epidermis in situ. TSH and TSH receptor (TSH-R) expression were analyzed in the epidermis of normal human scalp skin by immunohistochemistry and PCR. In addition, full-thickness scalp skin was organ cultured and treated with TSH, TRH, or thyroid hormones, and the effect of TSH treatment on the expression of selected genes was measured by quantitative PCR and/or quantitative immunohistochemistry. Here we show that normal human epidermis expresses TSH at the mRNA and protein levels in situ and transcribes TSH-R. It also contains thyrostimulin transcripts. Intraepidermal TSH immunoreactivity is up-regulated by TRH and down-regulated by thyroid hormones. Although TSH-R immunoreactivity in situ could not be documented within the epidermis, but in the immediately adjacent dermis, TSH treatment of organ-cultured human skin strongly up-regulated epidermal expression of involucrin, loricrin, and keratins 5 and 14. Thus, normal human epidermis in situ is both an extrapituitary source and (possibly an indirect) target of TSH signaling, which regulates defined epidermal parameters. Intraepidermal TSH expression appears to be regulated by the classical endocrine controls that determine the systemic HPT axis.

Advances in TERS (tip-enhanced Raman scattering) for biochemical applications

TERS (tip-enhanced Raman scattering) provides exceptional spatial resolution without any need for labelling and has become a versatile tool for biochemical analysis. Two examples will be highlighted here. On the one hand, TERS measurements on a single mitochondrion are discussed, monitoring the oxidation state of the central iron ion of cytochrome c, leading towards a single protein characterization scheme in a natural environment. On the other hand, a novel approach of single molecule analysis is discussed, again based on TERS experiments on DNA and RNA, further highlighting the resolution capabilities of this method.

Nestin in Human Skin: Exclusive Expression in Intramesenchymal Skin Compartments and Regulation by Leptin.

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.

Prolactin—a novel neuroendocrine regulator of human keratin expression in situ

The controls of human keratin expression in situ remain to be fully elucidated. Here, we have investigated the effects of the neurohormone prolactin (PRL) on keratin expression in a physiologically and clinically relevant test system: organ‐cultured normal human hair follicles (HFs). Not only do HFs express a wide range of keratins, but they are also a source and target of PRL. Microarray analysis revealed that PRL differentially regulated a defined subset of keratins and keratin‐associated proteins. Quantitative immunohistomorphometry and quantitative PCR confirmed that PRL up‐regulated expression of keratins K5 and K14 and the epithelial stem cell‐associated keratins K15 and K19 in organ‐cultured HFs and/or isolated HF keratinocytes. PRL also up‐regulated K15 promoter activity and K15 protein expression in situ, whereas it inhibited K6 and K31 expression. These regulatory effects were reversed by a pure competitive PRL receptor antagonist. Antagonist alone also modulated keratin expression, suggesting that “tonic stimulation” by endogenous PRL is required for normal expression levels of selected keratins. Therefore, our study identifies PRL as a major, clinically relevant, novel neuroendocrine regulator of both human keratin expression and human epithelial stem cell biology in situ. —Ramot, Y., Bíro´, T., Tiede, S. To´th, B. I., Langan, E. A., Sugawara, K., Foitzik, K., Ingber, A., Goffin, V., Langbein, L., Paus, R. Prolactin—a novel neuroendocrine regulator of human keratin expression in situ. FASEB J. 24, 1768–1779 (2010). www.fasebj.org

Basic fibroblast growth factor: a potential new therapeutic tool for the treatment of hypertrophic and keloid scars.

Numerous tissue niches in the human body, such as skin, are now recognized to harbour adult stem cells. In this study, we analyze multipotent human dermis-derived progenitor cell populations, isolated and propagated from mechanically and enzymatically processed adult scalp skin. The populations encompass Nestin-positive and -negative cells, which may serve as a convenient and abundant source for various therapeutic applications in regenerative medicine. Here, we show that these cultures exhibit a strong tendency to differentiate into mesodermal derivatives, particularly myofibroblasts, when maintained in media containing serum. Since undesired and excessive myofibroblast formation is a frequent postsurgical complication, we sought culture conditions that would prevent myofibroblast formation. In particular, we analyzed the effect of growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor AB (PDGF AB). Our results demonstrate that bFGF is a potent inhibitor of mesodermal differentiation, whereas PDFG AB favours myofibroblast formation and up-regulates expression of TGFbeta receptors I and II. This interesting discovery may help in the prevention and treatment of tissue fibrosis and in particular in the eradication of hypertrophic and keloid scars.