Generation of induced pluripotent stem cells (iPSCs) from human foreskin fibroblasts

The human iPS cell line VUZUZLi001-A (hVH-1) was generated from human foreskin fibroblasts to be used as a control line. Reprogramming was performed by retroviral transduction of reprogramming factors OCT4, SOX2, KLF4 and c-MYC.Resource tableUnlabelled TableUnique stem cell line identifierVUZUZLi001-AAlternative name(s) of stem cell linehVH-1InstitutionInstitute of Virology and Cell Biology, University of LübeckContact information of distributorJürgen Rohwedel, [email protected] of cell lineiPSCOriginhumanAdditional origin infoAge: unknownSex: maleEthnicity if known: —Cell sourceforeskin fibroblastsClonalitymixedMethod of reprogrammingretroviral integrationGenetic modificationNOType of modificationN/AAssociated diseaseN/AGene/locusN/AMethod of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock date2017-08-05Cell line repository/bankN/AEthical approvalEthics Committee University of Lübeck; reference number: 14-10

Murine mesenchymal progenitor cells from different tissues differentiated <it>via </it>mesenchymal microspheres into the mesodermal direction

Abstract Background Because specific marker molecules for phenotypical identification of mesenchymal stem and progenitor cells are missing, the assessment of the in vitro-differentiation capacity is a prerequisite to characterize these cells. However, classical differentiation protocols are often cell-consuming and time intensive. Therefore, the establishment of novel strategies for differentiation is one topic of current efforts in stem cell biology. The goal of this study was to demonstrate the practicability of a new differentiation test using plastic adherent cell isolates from different tissues. Results We introduced the mesenchymal microsphere method as a feasible time- and cell saving screening method to analyse multilineage differentiation properties of adult progenitor cells in a three-dimensional system. For this purpose we isolated, characterized and analyzed new sources of adult murine mesenchymal progenitor cells from perirenal adipose tissue and mediastinal stromal tissue in comparison to bone marrow progenitor cells. The proliferation capacity of the cells was demonstrated by determination of the daily doubling index. Although the flow cytometry analysis of undifferentiated cells revealed differences in the expression of CD marker molecules, all isolates have the capacity for multilineage differentiation following the mesenchymal microsphere protocol as well as the classical "micro mass body" protocol for chondrogenic and the monolayer cultivation protocol for osteogenic and adipogenic differentiation. Differentiation was characterized using histochemical and immunhistochemical staining as well as RT-PCR. Conclusions We were able to show that the mesenchymal microsphere method is an efficient test system for chondro-, osteo- and adipogenic differentiation of adult progenitor cells. The advantage of this system in comparison to classical protocols is that approximately 7 times lower cell numbers are necessary. Since classical culture procedures are time intensive because high cell numbers have to be obtained, the new differentiation method may also save cells and time in future clinical applications using human mesenchymal stromal cells.</p

Enhanced fibrillin-2 expression is a general feature of wound healing and sclerosis: potential alteration of cell attachment and storage of TGF-beta

Wound healing and sclerosis are characterized by an increase of extracellular matrix proteins, which are characteristically expressed in the embryo–fetal period. We analyzed the expression of fibrillin-2, which is typically found in embryonic tissues, but only scarcely in adult skin. In wound healing and sclerotic skin diseases such as lipodermatosclerosis and scleroderma, a marked increase of fibrillin-2 expression was found by immunohistology. Double labelling of fibrillin-2 and tenascin-C, which is also expressed in wound healing and sclerosis, showed co-localization of both proteins. Solid-phase and slot blot-overlay assays showed a dose-dependent binding of the recombinant N-terminal half of fibrillin-2 (rFBN2-N) to tenascin-C. Real-time PCR showed an increase of the fibrillin-2 gene expression in cell culture triggered by typical mediators for fibroblast activation such as serum, IL-4, and TGF-β. By contrast, prolonged hypoxia is not associated with changes in fibrillin-2 expression. Tenascin-C is an anti-adhesive substrate for fibroblasts, whereas fibrillin-2 stimulates cell attachment. Attachment assays using mixed substrates showed decreased cell attachment when tenascin-C and rFBN2-N were coated together, compared with the attachment to rFBN2-N alone. Fibrillins are involved in storage and activation of TGF-β. Immunohistology with an antibody against the latency-associated peptide (LAP (TGF-β1)) showed a marked increase of inactive LAP-bound TGF-β1 in wound healing and sclerotic skin whereas normal skin showed only a weak expression. Double immunofluorescence confirmed a partial colocalization of both proteins. In conclusion, we show that a stimulation of the fibrillin-2 expression is a characteristic feature of fibroblasts present in wound healing and sclerosis, which may be involved in the alteration of cell attachment and storage of inactive TGF-β in the matrix.Jürgen Brinckmann, Nico Hunzelmann, Birgit Kahle, Jürgen Rohwedel, Jan Kramer, Mark A Gibson, Dirk Hubmacher and Dieter P Reinhard