Hepatocyte growth factor-loaded biomaterials for mesenchymal stem cell recruitment.

Human adult mesenchymal stem cells (MSC) can be readily harvested from bone marrow through aspiration. MSC are involved in tissue regeneration and repair, particularly in wound healing. Due to their high self-renewal capacity and excellent differentiation potential in vitro, MSC are ideally suited for regenerative medicine. The complex interactions of MSC with their environment and their influence on the molecular and functional levels are widely studied but not completely understood. MSC secrete, for example, hepatocyte growth factor (HGF), whose concentration is enhanced in wounded areas and which is shown to act as a chemoattractant for MSC. We produced HGF-loaded biomaterials based on collagen and fibrin gels to develop a recruitment system for endogenous MSC to improve wound healing. Here, we report that HGF incorporated into collagen or fibrin gels leads to enhanced and directed MSC migration in vitro. HGF-loaded biomaterials might be potentially used as in vivo wound dressings to recruit endogenous MSC from tissue-specific niches towards the wounded area. This novel approach may help to reduce costly multistep procedures of cell isolation, in vitro culture, and transplantation usually used in tissue engineering.

Fluorescent SNAP-tag galectin fusion proteins as novel tools in glycobiology.

Galectins,ß-galactoside binding proteins, function in several physiological and pathological processes. The further evaluation of these processes as well as possible applications of galectins in diagnosis and therapy has raised high scientific interest. Therefore, easy and reliable test systems are necessary. Here we present the simple and cost-efficient production of recombinant human galectins as fusion proteins with SNAP-tag and fluorescent proteins. These constructs show binding specificities and oligomerisation properties generally comparable to recombinant galectins. Their direct fluorescence signal was utilised by ELISA-type assay and flow cytometry analysis with human and ovine mesenchymal stem cells (MSC). Flow cytometry demonstrated glycan mediated binding of His6-SNAP-YFP-Gal- 3 to both MSC types, which was specifically inhibited by lactose. Moreover, directed immobilisation by SNAP-tag technology onto benzylguanine- activated sepharose was utilised to prepare galectin affinity columns for glycoprotein analysis and purification. The SNAPtag directed coupling yielded up to three-fold higher binding capacities for the glycoprotein standard asialofetuin compared to nondirected coupled galectin suggesting improved functionality following directed coupling.

Epithelial morphogenesis of germline-derived pluripotent stem cells on organotypic skin equivalents in vitro.

For tissue engineering, cultivation of pluripotent stem cells on three-dimensional scaffolds allows the generation of organ-like structures. Previously, we have established an organotypic culture system of skin to induce epidermal differentiation in adult stem cells. Multipotent stem cells are not able to differentiate across germinal boundaries. In contrast, pluripotent stem cells readily differentiate into tissues of all three germ layers. Germline-derived pluripotent stem cells (gPS cells) can be generated by induction of pluripotency in mouse unipotent germline stem cells without the introduction of exogenous transcription factors. In the current study, we analyzed the influence of organotypic culture conditions of skin on the epithelial differentiation of gPS cells in comparison to the well-established HM1 ES cell line. Quantitative RT-PCR data of the pluripotency gene Oct4 showed that gPS cells are characterized by an accelerated Oct4-downregulation compared to HM1 ES cells. When subjected to the organotypic culture conditions of skin, gPS cells formed tubulocystic structures lined by stratified (CK5/6(+), CK14(+), CK8/18(-)) epithelia. HM1 ES cells formed only small tubulocystic structures lined by simple, CK8/18(+) epithelia. BMP-4, an epidermal morphogen, significantly enhanced the expression of epithelial markers in HM1 ES cells, but did not significantly affect the formation of complex (squamous) epithelia in gPS cells. In HM1 ES cells the differentiation into squamous epithelium was only inducible in the presence of mature dermal fibroblasts. Both pluripotent stem cell types spontaneously differentiated into mesodermal, endodermal and into neuroectodermal cells at low frequency, underlining their pluripotent differentiation capacity. Concluding, the organotypic culture conditions of skin induce a multilayered, stratified epithelium in gPS cells, in HM1 ES cells only in the presence of dermal fibroblasts. Thus, our data show that differentiation protocols strongly depend on the stem cell type and have to be modified for each specific stem cell type.