An innervated skin 3D in vitro model for dermatological research

A 3D in vitro model of innervated skin would be a useful tool in dermatological research to study the effect of different chemicals and compounds on the sensory properties of skin. Current innervated skin models are limited in composition and often composed of ex vivo skin explants and/or animal-derived material. In this study, our aim was to develop a human innervated skin model with a better biomimicry composition for in vitro research. Fibrin hydrogel and aligned electrospun fibers of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) were used as a scaffold to generate the 3D in vitro model. The skin component was made of primary human keratinocytes and primary human fibroblasts, while the neuronal component was composed of iPSC-derived sensory neurons. Our results showed that the dermal component consisted of fibroblasts and synthesized collagen. The epidermal component was characterized by the expression of keratins 10 and 14, and involucrin. Finally, sensory neurons extended axons throughout the scaffold and reached the epidermis. Treating the model with a capsaicin solution for 30 min, which was performed as a proof of concept test for sensitization studies, resulted into partial depletion of substance P and tubulin β3. This model could be used for studying skin-neuron interactions and cutaneous toxicity.</p

Neue Marker für prospektive Isolierung und Charakterisierung von Stromazellen, Endothelzellen und Spermatogonien, Vorläuferzellen aus adulten menschlichen Hoden

The isolation of spermatogonia from adult human testis is hampered by the limited selectivity of available markers. In this study, I evaluated the suitability of combinations of known markers and novel targets for the prospective isolation of spermatogonia from adult human testis. Immunohistochemical studies, multicolor staining and cell sorting of testis samples followed by multiplex PCR and dendrogram cluster analysis revealed that spermatogonia were enriched at high purity in the CD49f+CD49a-SSEA-4+SUSD2+ population as well as in the CD49f+CD49a-SSEA-4+/-CD164+ subsets. In contrast to spermatogonia, testicular stromal cells and testicular endothelial cells were found to be highly enriched in the CD49f+CD49a+CD144- and in the CD49f+CD49a+CD144+ subsets, respectively. The delineation was confirmed by the expression of specific stromal and endothelial key markers as well as by the differentiation capacity of each subset. In addition, I identified for the first time two distinct subsets of spermatogonia, distinguished by their CD49f+SSEA-4+CD164+ and CD49f+SSEA-4-CD164+ expression profiles. Multiplex PCR analysis revealed that cells of the CD49f+SSEA-4-CD164+ subset express spermatogonia/germ cell specific markers, whereas the CD49f+SSEA-4+CD164+ subset is enriched in undifferentiated spermatogonia specific markers. The knowledge about the composite phenotype of defined testicular cell subsets and the reliable isolation procedure may contribute to the precise characterization and efficient isolation of spermatogonia from small biopsies and may contribute to the treatment of infertility.Die Isolierung von Spermatogonien aus adulten menschlichen Hoden ist zur Zeit wegen der begrenzten Zahl und dem Mangel an selektiven Markern nur eingeschränkt möglich. In der vorliegenden Arbeit wurden Kombinationen bekannter Marker und neuer Targets, die für die Isolierung von Spermatogonien geeignet sind, untersucht. Mit Hilfe immunhistochemischer Analysen, Mehrfarbenfluoreszenzanalysen und Zellsortierung von Proben aus Hodengewebe sowie nachfolgenden Multiplex-PCR und Dendrogramm Clusteranalysen konnte gezeigt werden, dass Spermatogonien sowohl in der CD49f+CD49a-SSEA-4+SUSD2+ Population als auch im CD49f+CD49a-SSEA-4+/-CD164+ Subset hochrein isoliert werden können. Außer Spermatogonien konnten aus Hodengewebe auch Stromazellen in der CD49f+CD49a+CD144- Population und Endothelzellen im CD49f+CD49a+CD144+ Subset isoliert werden. Die Unterscheidung von Endothelzellen und Stromazellen wurde über den Nachweis der spezifischen Expression von Schlüsselmarkern sowie durch die unterschiedliche Differenzierungkapazität der jeweiligen Zellpopulationen erzielt. Darüber hinaus konnten zum ersten Mal zwei unterschiedliche Populationen von Spermatogonien identifiziert werden, die sich durch ihre differenziellen Expressionsprofile unterscheiden. So konnte über Multiplex PCR-Analysen gezeigt werden, dass CD49f+SSEA-4-CD164+ Zellen Keimzell-spezifische Marker exprimierten, die einem differenzierteren Stadium von Spermatogonien entsprechen, während Zellen im CD49f+SSEA-4+CD164+ Subset Marker exprimierten, die typischerweise bei Spermatogonien im undifferenzierten Stadium vorkommen. Die Kenntnis des genauen Phänotyps von Spermatogonien sowie die hochreine Isolierung dieser seltenen Zellpopulation ist möglicherweise ein wichtiger Baustein bei der Behandlung von Unfruchtbarkeit

Development of an In Vitro Biomimetic Peripheral Neurovascular Platform

[Image: see text] Nerves and blood vessels are present in most organs and are indispensable for their function and homeostasis. Within these organs, neurovascular (NV) tissue forms congruent patterns and establishes vital interactions. Several human pathologies, including diabetes type II, produce NV disruptions with serious consequences that are complicated to study using animal models. Complex in vitro organ platforms, with neural and vascular supply, allow the investigation of such interactions, whether in a normal or pathological context, in an affordable, simple, and direct manner. To date, a few in vitro models contain NV tissue, and most strategies report models with nonbiomimetic representations of the native environment. To this end, we have established here an NV platform that contains mature vasculature and neural tissue, composed of human microvascular endothelial cells (HMVECs), induced pluripotent stem cell (iPSCs)-derived sensory neurons, and primary rat Schwann cells (SCs) within a fibrin-embedded polymeric scaffold. First, we show that SCs can induce the formation of and stabilize vascular networks to the same degree as the traditional and more thoroughly studied human dermal fibroblasts (HDFs). We also show that through SC prepatterning, we are able to control vessel orientation. Using our NV platform, we demonstrate the concomitant formation of three-dimensional neural and vascular tissue, and the influence of different medium formulations and cell types on the NV tissue outcome. Finally, we propose a protocol to form mature NV tissue, via the integration of independent neural and vascular constituents. The platform described here provides a versatile and advanced model for in vitro research of the NV axis

Decellularization of porcine heart tissue to obtain extracellular matrix based hydrogels

The use of hydrogels derived from the extracellular matrix (ECM) in tissue engineering applications aims to overcome the conundrum of mimicking the complexity of ECM composition in vitro. In this chapter, we describe a method of decellularization and subsequent formation of an ECM-based hydrogel using porcine heart tissue. These decellularized ECM hydrogels could be used to create semi-interpenetrating networks or as bioinks in bioprinting applications to further enhance the bioactivity and increase the biomimicry degree of the biological cardiac constructs

Decellularization of porcine heart tissue to obtain extracellular matrix based hydrogels

The use of hydrogels derived from the extracellular matrix (ECM) in tissue engineering applications aims to overcome the conundrum of mimicking the complexity of ECM composition in vitro. In this chapter, we describe a method of decellularization and subsequent formation of an ECM-based hydrogel using porcine heart tissue. These decellularized ECM hydrogels could be used to create semi-interpenetrating networks or as bioinks in bioprinting applications to further enhance the bioactivity and increase the biomimicry degree of the biological cardiac constructs