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The healing of neuronal injuries is still an unachieved goal. Medicine-based therapies can only extend the survival of patients, but not finally lead to a healing process. Currently, a variety of stem cell-based tissue engineering developments are the subject of many research projects to bridge this gap. As yet, neuronal differentiation of induced pluripotent stem cells (iPS), embryonic cell lines, or neuronal stem cells could be accomplished and produce functional neuronally differentiated cells. However, clinical application of cells from these sources is hampered by ethical considerations. To overcome these hurdles numerous studies investigated the potential of adult mesenchymal stem cells (MSCs) as a potential stem cell source. Adult MSCs have been approved as cellular therapeutical products due to their regenerative potential and immunomodulatory properties. Only a few of these studies could demonstrate the capacity to differentiate MSCs into active firing neuron like cells. With this study we investigated the potential of Wharton’s Jelly (WJ) derived stem cells and focused on the intrinsic pluripotent stem cell pool and their potential to differentiate into active neurons. With a comprehensive neuronal differentiation protocol comprised of mechanical and biochemical inductive cues, we investigated the capacity of spontaneously forming stem cell spheroids (SCS) from cultured WJ stromal cells in regard to their neuronal differentiation potential and compared them to undifferentiated spheroids or adherent MSCs. Spontaneously formed SCSs show pluripotent and neuroectodermal lineage markers, meeting the pre-condition for neuronal differentiation and contain a higher amount of cells which can be differentiated into cells whose functional phenotypes in calcium and voltage responsive electrical activity are similar to neurons. In conclusion we show that up-concentration of stem cells from WJ with pluripotent characteristics is a tool to generate neuronal cell replacement. Graphical Abstract

PURPOSE A malfunction of the corneal endothelium leading to corneal opacity is one of the main causes of impaired vision. Currently, keratoplasty is the one and only donor cornea-dependent treatment, and this calls for alternatives because of the worldwide lack of donor corneas. Recently, the topography of Descemet membrane (DM) has been discovered as a feasible stem cell differentiation tool. With this study, we further confirm this mechanotransductive system by using preinduced Wharton jelly-derived mesenchymal stem cells (WJ-EPCs). METHODS To measure the mechanotransductive potential of Descemet-like topography (DLT), WJ-EPCs were cultivated on collagen imprints with DLT. Changes in the gene and protein expressions of corneal endothelial cells (CECs), typical markers such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured. In addition, CEC functionality has been evaluated by measuring the relative potential differences in a 2-compartment system and by measuring corneal transparency in an ex vivo rabbit cornea model. To confirm the activity of WJ-EPCs, rabbit CECs were restless deleted by collagen digestion of a thin layer of rabbit Descemet membrane. RESULTS The proper CEC-typical hexagonal morphology of WJ-EPCs in combination with a significant expression of ZO-1, Na/K-ATPase, PITX2, and COL-8 could be demonstrated. In addition, the WJ-EPCs were able to build up a relative potential difference of 40 mV and to keep corneas clear and transparent. CONCLUSIONS These data indicate that a well-characterized, functional CEC monolayer was developed by using a DLT-mediated mechanotransductive differentiation of WJ-EPCs.

Corneal endothelial insufficiency is one of the leading causes of blindness. The main contemporary treatment for corneal blindness is endothelial keratoplasty, which, however, is unsatisfactory as a medical therapy due to the lack of donor corneas and graft rejection. Therefore, autologous stem cell-based corneal endothelial tissue substitutes may be a promising alternative to conventional grafts in the future. To address the age of most patients suffering from corneal endothelial deficiencies, we investigated the presence and potential of hair-derived stem cells from older tissue donors. Our studies revealed the presence of pluripotency- and neural crest-associated markers in tissue sections from blepharoplasty patients aged 50 to 80 years. In vitro outgrowths from eyelid hair follicles on collagen-coated tissue culture plates revealed a weak decrease in stem-cell potency. In contrast, cells within the spheres that spontaneously formed from the adherent cell layer retained full stem-cell potency and could be differentiated into cells of the ecto- meso and endodermal lineages. Although these highly potent hair follicle derived stem cells (HFSC) were only very slightly expandable, they were able to recognize the biomimicry of the Descemet’s-like topography and differentiate into corneal endothelial-like cells. In conclusion, HFSCs derived from epidermal skin of eyelid biopsies are a promising cell source to provide autologous corneal endothelial replacement for any age group of patients. Graphical Abstract

Supplemental Digital Content is Available in the Text., Purpose: Loss of corneal endothelial cells (CECs) bears disastrous consequences for the patient, including corneal clouding and blindness. Corneal transplantation is currently the only therapy for severe corneal disorders. However, the worldwide shortages of corneal donor material generate a strong demand for personalized stem cell–based alternative therapies. Because human mesenchymal stem cells are known to be sensitive to their mechanical environments, we investigated the mechanotransductive potential of Descemet membrane–like microtopography (DLT) to differentiate human mesenchymal stem cells into CEC-like cells. Methods: Master molds with inverted DLT were produced by 2-photon lithography (2-PL). To measure the mechanotransductive potential of DLT, mesenchymal stem cells were cultivated on silicone or collagen imprints with DLT. Changes in morphology were imaged, and changes in gene expression of CEC typical genes such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured with real-time polymerase chain reaction. At least immunofluorescence analysis has been conducted to confirm gene data on the protein level. Results: Adhesion of MSCs to DLT molded in silicone and particularly in collagen initiates polygonal morphology and monolayer formation and enhances not only transcription of CEC typical genes such as ZO-1, Na/K-ATPase, PITX2, and COL-8 but also expression of the corresponding proteins. Conclusions: Artificial reproduction of Descemet membrane with respect to topography and similar stiffness offers a potential innovative way to bioengineer a functional CEC monolayer from autologous stem cells.