Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog

Purpose: Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis formation and stratification in a humanized animal model. Methods: Dermo-epidermal skin grafts with either amniocytes or with fibroblasts in the dermis were compared in a rat model. Full-thickness skin wounds on the back of immuno-incompetent rats were covered with skin grafts with (1) amniocytes in the dermis, (2) fibroblasts in the dermis, or, (3) acellular dermis. Grafts were excised 7 and 21days post transplantation. Histology and immunofluorescence were performed to investigate epidermis formation, stratification, and expression of established skin markers. Results: The epidermis of skin grafts engineered with amniocytes showed near-normal anatomy, a continuous basal lamina, and a stratum corneum. Expression patterns for keratin 15, keratin 16, and Ki67 were similar to grafts with fibroblasts; keratin 1 expression was not yet fully established in all suprabasal cell layers, expression of keratin 19 was increased and not only restricted to the basal cell layer as seen in grafts with fibroblasts. In grafts with acellular dermis, keratinocytes did not survive. Conclusion: Dermo-epidermal skin grafts with amniocytes show near-normal physiological behavior suggesting that amniocytes substitute fibroblast function to support the essential cross-talk between mesenchyme and epithelia needed for epidermal stratification. This novel finding has considerable implications regarding tissue engineerin

RADICL-seq identifies general and cell type–specific principles of genome-wide RNA-chromatin interactions

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure

The bioengineering of human blood and lymphatic capillaries in dermo-epidermal skin substitutes

Slow vascularization of bioengineered organs after transplantation constitutes a major hurdle in tissue engineering and regenerative medicine. Providing these organs with functional blood and lymphatic capillaries prior to transplantation (henceforth referred to as prevascularization) may be an attractive solution for this problem, given that the bioengineered capillaries rapidly connect to the recipient’s vasculature in the wound bed. Despite considerable progress in the bioengineering of functional blood capillaries, no data were available neither on the bioengineering of functional human lymphatic capillaries, nor on the integration of a dermal lymphatic network into a skin substitute. Moreover, it remained largely unknown how pre-existing blood and lymphatic capillaries affect the reconstitution of a dermo-epidermal skin substitute (DESS) after its transplantation. The first part of my work aimed at bioengineering prevascularized DESS (PDESS) containing physiologically distinct blood and lymphatic capillaries. For this, human dermal microvascular endothelial cells (HDMEC), dermal fibroblasts and epidermal keratinocytes isolated from human skin biopsies were sequentially seeded into and onto rapidly polymerizing fibrin hydrogels. Within 3 weeks of in vitro culture, lumenized and interconnected capillary networks consisting of blood and lymphatic capillaries spontaneously assembled within PDESS. I found that during the process of prevascularization, the inoculated fibroblasts differentiated into pericytes and specifically stabilized blood capillaries, while they hardly associated with lymphatic capillaries. The lymphatic capillaries presented anchoring filaments, expressed all major lymphatic markers, and could be modulated by lymphangiogenic and anti-lymphangiogenic stimuli. In addition, the lymphatic capillaries took up fluid from the interstitial space in vitro and improved the drainage of fluids from PDESS in vivo. Analysis of PDESS after transplantation revealed a great acceleration of vascular regeneration involving two distinct mechanisms: rapid anastomosis of the bioengineered blood capillaries - followed by lymphatic anastomosis - and accelerated recipient blood and lymphatic angiogenesis. In the second part of this work, I performed a broad analysis of the effects of prevascularization on several aspects of wound healing and skin morphogenesis of DESS. By comparison of PDESS and non-prevascularized DESS (NPDESS) in vivo, I found that the rapid perfusion of the bioengineered blood capillary network increased proliferation and decreased apoptosis of transplanted cells, reduced graft shrinkage, induced the formation of rete ridges and capillary loops and promoted dermal remodelling. Furthermore, PDESS healed by the deposition of randomized collagen bundles instead of the scar-like parallel collagen bundle orientation found after transplantation of NPDESS. Collectively, my data for the first time describe the bioengineering of clinically relevant PDESS containing human blood and lymphatic capillary networks that rapidly connect with the host vasculature after transplantation, thereby markedly accelerating perfusion of the graft. In vitro and in vivo studies indicate that the rapid perfusion of PDESS unlocks latent regenerative processes that shift the healing of DESS from typical scar formation towards regeneration. Die langsame Vaskularisierung künstlicher Organe stellt ein gravierendes Problem im Bereich Tissue engineering und Regenerative Medizin dar. Das Versetzen solcher Organe mit funktionalen Blut- und lymphatischen Gefässen vor der Transplantation (Prevascularization) repräsentiert eine attraktive Lösung für dieses Problem, bedingt jedoch, dass sich die künstlichen Gefässe nach der Transplantation schnell mit den Gefässen des Empfängers verbinden (Anastomose). Trotz bemerkenswerter Fortschritte in der Herstellung künstlicher Blutgefässe gab es bisher keine Berichte über die Herstellung von funktionalen lymphatischen Gefässen oder über die Versetzung von dermo- epidermalen Hautsubstituten (DESS) mit solchen lymphatischen Gefässen. Ausserdem war nur sehr wenig über den Effekt von konstruierten Blut- und lymphatischen Gefässen auf den Heilungsprozess eines dermo-epidermalen Hautersatzes bekannt. Das Ziel des ersten Teils meiner Arbeit war die Herstellung von prevaskularisierten dermo-epidermalen Hautsubstituten (PDESS), die künstliche Blut- und lymphatische Gefässe beinhalten. Dafür wurden human dermal microvascular endothelial cells (HDMEC), dermale Fibroblasten und epidermale Keratinocyten von einer Hautbiopsie isoliert und zuerst in und dann auf ein schnell polymerisierendes Fibrin Hydrogel gesäht. Innert 3 Wochen entstand so spontan ein Netzwerk verbundener Kapillaren mit Lumen, bestehend aus Blut- und lymphatischen Gefässen. Die Fibroblasten differenzierten während der Prevaskularisierung zu Perizyten und stabilisierten spezifisch die Blutkapillaren, während sie kaum mit lymphatischen Gefässen assoziierten. Die lymphatischen Gefässe entwickelten Anchoring filaments, exprimierten alle wichtigen lymphatischen Marker und konnten mittels lymphangiogenen und anti-lymphangiogenen Stimuli moduliert werden. Ausserdem waren sie fähig zur Flüssigkeitsaufnahme in vitro und verbesserten den Flüssigkeits-Abtransport in vivo. Die Analyse von PDESS nach der Transplantation hat gezeigt, dass Prevaskularisierung die vaskuläre Regeneration mittels zwei verschiedener Mechanismen beschleunigt: schnelle Anastomose der Blutgefässe – gefolgt von der Anastomose der lymphatischen Gefässe – und Beschleunigung des Einwuchses von Blut und lymphatischen Gefässen des Empfängers in das Hautsubstitut. Im zweiten Teil meiner Arbeit führte ich eine ausgiebige Analyse der Effekte von Prevaskularisierung auf die Heilung und Hautmorphogenese von PDESS durch. Mittels eines Vergleichs von PDESS und nicht-prevaskularisierten DESS (NPDESS) in vivo habe ich herausgefunden, dass die schnelle Perfusion der künstlichen Blutgefässe die Proliferation der transplantierten Zellen unterstützt und deren Apoptose vermindert, die Schrumpfung des Hautsubstituts reduziert, die Entwicklung von Rete ridges und Capillary Loops induziert und das dermale Remodelling fördert. Ausserdem heilten PDESS mittels Deposition von zufällig orientierten Kollagenbündeln anstatt von parallelen, Narben- Kollagenbündeln wie nach der Transplantation von NPDESS. Diese Arbeit beschreibt zum ersten Mal ein klinisch relevantes dermo-epidermales Hautsubstitut, welches sowohl Blut- als auch lymphatische Gefässe beinhaltet, durch deren schnelle Anastomose die Perfusion des Transplantats merklich beschleunigt wird. In vitro und in vivo Studien deuten darauf hin, dass die schnelle Perfusion ruhende regenerative Prozesse in PDESS aktiviert, welche den Heilungsprozess von einer typischen Narbenformation in Richtung Regeneration leiten

The essentiality of non-coding RNAs in cell reprogramming

In mammals, short (mi-) and long non-coding (lnc) RNAs are immensely abundant and they are proving to be more functional than ever before. Particularly in cell reprogramming, non-coding RNAs are essential to establish the pluripotent network and are indispensable to reprogram somatic cells to pluripotency. Through systematic screening and mechanistic studies, diverse functional features of both miRNA and lncRNAs have emerged as either scaffolds, inhibitors, or co-activators, necessary to orchestrate the intricacy of gene regulation. Furthermore, the collective characterizations of both miRNA and lncRNA reveal their interdependency (e.g. sequestering the function of the other) to modulate cell reprogramming. This review broadly explores the regulatory processes of cell reprogramming - with key functional examples in neuronal and cardiac differentiations - in the context of both short and long non-coding RNAs. Keywords: miRNA, lncRNA, Reprogramming, Pluripotency, Neuron

Modified plastic compression of collagen hydrogels provides an ideal matrix for clinically applicable skin substitutes

Tissue engineering of clinically applicable dermo-epidermal skin substitutes is crucially dependent on the three-dimensional extracellular matrix, supporting the biological function of epidermal and dermal cells. This matrix essentially determines the mechanical stability of these substitutes to allow for safe and convenient surgical handling. Collagen type I hydrogels yield excellent biological functionality but their mechanical weakness and their tendency to contract and degrade does not allow producing clinically applicable transplants of larger sizes. We show here that plastically compressed collagen type I hydrogels can be produced in clinically relevant sizes (7 x 7 cm), and can be safely and conveniently handled by the surgeon. Most importantly, these dermo-epidermal skin substitutes mature into a near normal skin that can successfully reconstitute full thickness skin defects in an animal model

The accuracy of FAST in relation to grade of solid organ injuries: A retrospective analysis of 226 trauma patients with liver or splenic lesion

Abstract Background This study investigated the role of a negative FAST in the diagnostic and therapeutic algorithm of multiply injured patients with liver or splenic lesions. Methods A retrospective analysis of 226 multiply injured patients with liver or splenic lesions treated at Bern University Hospital, Switzerland. Results FAST failed to detect free fluid or organ lesions in 45 of 226 patients with spleen or liver injuries (sensitivity 80.1%). Overall specificity was 99.5%. The positive and negative predictive values were 99.4% and 83.3%. The overall likelihood ratios for a positive and negative FAST were 160.2 and 0.2. Grade III-V organ lesions were detected more frequently than grade I and II lesions. Without the additional diagnostic accuracy of a CT scan, the mean ISS of the FAST-false-negative patients would be significantly underestimated and 7 previously unsuspected intra-abdominal injuries would have been missed. Conclusion FAST is an expedient tool for the primary assessment of polytraumatized patients to rule out high grade intra-abdominal injuries. However, the low overall diagnostic sensitivity of FAST may lead to underestimated injury patterns and delayed complications may occur. Hence, in hemodynamically stable patients with abdominal trauma, an early CT scan should be considered and one must be aware of the potential shortcomings of a "negative FAST".</p

Tissue-engineered dermo-epidermal skin grafts prevascularized with adipose-derived cells

The major problem in skin grafting is that tissue-engineered skin grafts after their transplantation are initially entirely dependent on diffusion. Since this process is slow and inefficient, nutrients, growth factors, and oxygen will insufficiently be supplied and the regenerating graft will undergo a physiological crisis, resulting in scar-like dermal structures and shrinkage. The tissue-engineering of a vascular network in human dermo-epidermal skin substitutes (DESS) is a promising approach to overcome this limitation. Here we report, for the first time, on the use of the adipose stromal vascular fraction (SVF)-derived endothelial cell population to tissue-engineer DESS containing a highly efficient capillary plexus. To develop vascular networks in vitro, we employed optimized 3D fibrin or collagen type I hydrogel systems. Upon transplantation onto immune-deficient rats, these pre-formed vascular networks anastomosed to the recipient's vasculature within only four days. As a consequence, the neo-epidermis efficiently established tissue homeostasis, the dermis underwent almost no contraction, and showed sustained epidermal coverage in vivo. Overall, the here described rapid and efficient perfusion of SVF-based skin grafts opens new perspectives for the treatment of hitherto unmet clinical needs in burn/plastic surgery and dermatology

Decoding Neuronal Diversification by Multiplexed Single-cell RNA-Seq

Cellular reprogramming is driven by a defined set of transcription factors; however, the regulatory logic that underlies cell-type specification and diversification remains elusive. Single-cell RNA-seq provides unprecedented coverage to measure dynamic molecular changes at the single-cell resolution. Here, we multiplex and ectopically express 20 pro-neuronal transcription factors in human dermal fibroblasts and demonstrate a widespread diversification of neurons based on cell morphology and canonical neuronal marker expressions. Single-cell RNA-seq analysis reveals diverse and distinct neuronal subtypes, including reprogramming processes that strongly correlate with the developing brain. Gene mapping of 20 exogenous pro-neuronal transcription factors further unveiled key determinants responsible for neuronal lineage specification and a regulatory logic dictating neuronal diversification, including glutamatergic and cholinergic neurons. The multiplex scRNA-seq approach is a robust and scalable approach to elucidate lineage and cellular specification across various biological systems.ISSN:2213-671