Search

Die Homöostase wird maßgeblich durch die Niere, bestehend aus Millionen funktioneller Untereinheiten, den Nephronen, aufrechtherhalten. Chronisch geschädigte Nephrone führen zur Entwicklung einer terminalen Nierenerkrankung (TNE). Die Erzeugung renaler Zellen aus humanen pluripotenten Stammzellen (hPSCs) stellt eine vielversprechende Strategie zur regenerativen Therapie und Behandlung von TNE dar. In der vorliegenden Arbeit wurde ein Protokoll zur Differenzierung von renalen Vorläufern (RV) aus hPSCs entwickelt, welches nephronale Zelltypen und Strukturen in vitro und ex vivo erzeugte. Eine selektierte Kombination von Faktoren wurde in diesem 8-Tage-Protokoll genutzt, um die schrittweise Differenzierung der hPSCs zu lenken, indem die embryonale Organogenese der Niere abgebildet wurde. Am Tag 6 der Differenzierung konnten SIX2+/CITED1+ Zellen des metanephrischen Mesenchyms und HOXB7+/GRHL2+ Zellen, welche auf Vorläufer der Ureterknospe hindeuten, nachgewiesen werden. Diese entwickelten sich am Tag 8 weiter zu LGR5+/JAG1+/WT1+ renalen Vesikelzellen. Weiterführende Kultivierung in drei verschiedenen induktiven Medien führte zu WT1+/PODXL+/SYNPO+ Podozytenvorläufern, PDGFRß+/DESMIN+/αSMA+ Mesangialzellen und epithelialen Zellen des proximalen und distalen Tubulus sowie des Sammelrohrs. Außerdem bildeten die Tag-8-Vorläuferzellen spontan 3D renale Organoide aus. Die RV induzierten tubuläre Strukturen an einer Luft-Flüssigkeits-Grenzfläche und integrierten sich in embryonale Nierenaggregate. Zusammenfassend konnte demnach ein Protokoll entwickelt werden, welches entstehenden Nephronen ähnliche RV generierte, die innerhalb von 14 Tagen in spezialisierte nephronale Zelltypen differenzierten. Diese einfache Methode, um renale Zellen aus einem gemeinsamen Vorläuferpool in einer 2D -Kultur zu erzeugen, schafft die Grundlage für eine Produktion im größeren Maßstab, sowie für Modellsysteme in toxikologischen Untersuchungen oder Zelltherapien., Kidneys are the central organ for homeostasis for our body systems and composed of around a million functional units, the nephrons. Chronically damaged nephrons deteriorate progressively towards end stage renal disease (ESRD), owing to the limited regenerative capacity of adult mammalian kidneys. The generation of renal cells from human pluripotent stem cells (hPSCs) is a promising strategy to develop regenerative therapies for ESRD. In this study, we established a protocol to differentiate hPSCs to renal progenitors (RP), capable of producing nephronal cell types and structures in vitro and ex vivo. An effective combination of factors obtained after intensive screening, was used to create an 8-day-protocol that steered hPSCs to the renal lineage by a step-wise process outlining the embryonic milestones in kidney organogenesis. Six days after growth factor treatment, a mixture of SIX2+/CITED1+ cells representing metanephric mesenchyme and an HOXB7+/GRHL2+ population indicative of ureteric bud progenitors was obtained that developed into LGR5+/JAG1+/WT1+ renal vesicle cells by the day 8. Prolonged cultivation of these day 8 cells in three inductive media resulted in generation of WT1+/PODXL+/SYNPO+ podocyte-precursors, PDGFRß+/DESMIN+/αSMA+-mesangial cells and fractions of proximal, distal and collecting duct tubular epithelial cells in vitro. Moreover, day 8 cells differentiate spontaneously into renal organoids in culture. The hPSC-derived RP gave rise to tubular structures upon culture as a pellet in air-liquid interface and integrated into embryonic kidney re-aggregations. Thus, we demonstrate that our protocol generates RP reminiscent of nascent nephrons, which can be coaxed into specialized nephronal cell types in vitro after 14 days from hPSCs. This simple and rapid method to produce renal cells from a common precursor pool in 2D culture provides the basis for scaled-up production of tailored renal cell types, applicable for drug testing or cell therapies.

The human skin is involved in protecting the inner body from constant exposure to outer environmental stimuli. There is an evident need to screen for toxicity and the efficacy of drugs and cosmetics applied to the skin. To date, animal studies are still the standard method for substance testing, although they are currently controversially discussed Therefore, the multi-organ chip is an attractive alternative to replace animal testing. The two-organ chip is designed to hold 96-well cell culture inserts (CCIs). Small-sized skin equivalents are needed for this. In this study, full-thickness skin equivalents (ftSEs) were generated successfully inside 96-well CCIs. These skin equivalents developed with in vivo-like histological architecture, with normal differentiation marker expressions and proliferation rates. The 96-well CCI-based ftSEs were successfully integrated into the two-organ chip. The permeation of fluorescein sodium salt through the ftSEs was monitored during the culture. The results show a decreasing value for the permeation over time, which seems a promising method to track the development of the ftSEs. Additionally, the permeation was implemented in a computational fluid dynamics simulation, as a tool to predict results in long-term experiments. The advantage of these ftSEs is the reduced need for cells and substances, which makes them more suitable for high throughput assays., The human skin is involved in protecting the inner body from constant exposure to outer environmental stimuli. There is an evident need to screen for toxicity and the efficacy of drugs and cosmetics applied to the skin. To date, animal studies are still the standard method for substance testing, although they are currently controversially discussed Therefore, the multi-organ chip is an attractive alternative to replace animal testing. The two-organ chip is designed to hold 96-well cell culture inserts (CCIs). Small-sized skin equivalents are needed for this. In this study, full-thickness skin equivalents (ftSEs) were generated successfully inside 96-well CCIs. These skin equivalents developed with in vivo-like histological architecture, with normal differentiation marker expressions and proliferation rates. The 96-well CCI-based ftSEs were successfully integrated into the two-organ chip. The permeation of fluorescein sodium salt through the ftSEs was monitored during the culture. The results show a decreasing value for the permeation over time, which seems a promising method to track the development of the ftSEs. Additionally, the permeation was implemented in a computational fluid dynamics simulation, as a tool to predict results in long-term experiments. The advantage of these ftSEs is the reduced need for cells and substances, which makes them more suitable for high throughput assays.