immunofluorescence staining of organotypic slices with stem cell transplants for confocal microscopy

Langzeittransplantation von h-SC-NES-Zellen in organotypische Kulturen des Rückenmarks

Traumatic spinal cord injury (SCI) has devastating physical, psychological, and economic consequences for patients and caregivers1. Many attempts have been made to promote axonal regeneration in SCI with different approaches2,3,4 and some beneficial effects were demonstrated by the formation of neuronal relays between proximal and distal neurons in the injury site through cell replacement therapies. The interest in cell therapies is still growing5 since transplanted cells can play many roles, including providing trophic support, immune modulation, regenerating lost neural circuits through induction of plasticity, cell replacement, and axon remyelination6.
Recently, the main effort in the field focused on human neural stem/progenitor cells (NSCs/NPCs)7. Several studies suggest that NSCs/NPCs modulate astrocyte response8, promote the secretion of proregenerative factors9,10, and replace missing neuronal cells in SCI11,12. However, studies that support the differentiation of transplanted cells into functional neurons are still poor. Moreover, transplanted cell survival and differentiation in the injured spinal cord (SC) are low13, possibly because transplanted cells take several weeks, even months, to differentiate in vivo. Additionally, current studies have not completely elucidated many biochemical, molecular, cellular, and functional aspects of cell replacement therapies. In this context, simple, fast, and cost-effective models are required to study the mechanisms of cell engraftment, the ability of engrafted cells to proliferate, differentiate into specific types or subpopulations of cells, and form synapses with resident neurons.
Integrating histological studies into electrophysiological recording and transcriptome and proteome profiling is necessary for a full comprehension of the molecular cascade occurring after cell transplantation. This certainly will speed up the design and validation of new cell replacement therapies in preclinical models and clinical studies. Indeed, to date, the use of rodents, large animals, and non-human primates has been worthwhile for elucidating many cellular processes after transplantation14. However, due to the high cost, the high ethical impact, as well as the complexity of the organism, their use is often not straightforward or not adequate to unravel biochemical and molecular processes. In addition, they may present many disadvantages correlated with biological differences, both interspecies (metabolism) and intraspecies variability (sex, age).&#160;&#160;These factors, together with external factors such as stressful situations, could alter the outcome of an experiment and their predictability in terms of therapeutic translation to humans15,16,17.
Thus, many groups employ 2D in vitro cell culture and ex vivo organotypic slices (ex vivo cultures) in addition to animal models. 2D cell culture is the most commonly used system for studying specific biological processes at a single-cell and/or cell population level. Nevertheless, monolayer cell cultures do not reflect the complexity found in a whole organism. The lack of tissue structures and&#160;physiological environment does not allow the 2D culture systems to completely emulate key structural, morphological, and functional aspects of the investigated tissue18,19,20. Organotypic cultures can overcome some of these issues. Organotypic models are based on explanting a fragment of a tissue or organ and maintaining it ex vivo for a limited period21,22. In particular, slices of the explanted tissue are generated with a precise thickness that allows nutrients to easily reach almost all the cells in the slices. They&#160;can be generated from various regions of the central nervous system, such as the hippocampus, hypothalamus, cerebellum, thalamus, cerebral cortex, substantia nigra and striatum, and spinal cord23. Organotypic cultures retain the tissue architecture, the spatial distribution of cells, the cellular diversity, and the environment (i.e., extracellular matrix composition) of the organ of origin. Moreover, they preserve the original neural activity, connections between cells, and in particular, short-distance circuits after the explant.
These aspects provide some advantages for ex vivo cultures with respect to both monolayer cultures and animal models. They retain key tissue features found in vivo but with the reduction of the costs and the possibility to perform different types of molecular, cellular, and functional experiments with an accurate regulation of the culture environmental parameters24,25,26,27,28,29. Organotypic slices can also be exploited to develop models for different neurological disorders by resembling key histopathological features of specific conditions30. Moreover, the retention of the original multicellular tissue environment renders them appropriate platforms for drug screening and for testing neuroprotective and neuro-regenerative molecules and materials.
In this work, we propose the use of SC organotypic cultures as a model to optimize NSC transplants. This is not trivial since optimal culturing conditions are required to guarantee the survival of both the host (SC tissue) and the transplant (NSCs) for weeks. Different research groups engrafted in organotypic cultures, brain-derived and SC-derived, various types of cells. Most of the works showed the transplantation of mesenchymal stem cells31,32,33, olfactory ensheathing cells34, or NSCs35,36,37,38,39,40 and evaluated the interactions of engrafted cells with the host cells, the survival of the whole system, and whether the transplanted cells differentiated into neurons or neuron-like cells inside the ex vivo tissue environment32,33,41. Some of them evaluated the regenerative potential of cells after transplant, observing their axonal growth inside the tissue37,40,41, the myelinating ability of engrafted precursors of oligodendrocytes42, the migration of engrafted cells into the host tissue43, and whether the transplanted cells released factors pushing towards a proregenerative environment31. One limitation of the current studies is that they do not explore the engraftment for a long-term period.
Considering that NSCs seem to require several weeks to differentiate in vivo44,45, this study focuses on how to generate and maintain long-term (&#8805;30 days) mouse SC slices for up to 90 days. Slices were found to retain their original anatomical structure and to maintain a low and stable apoptotic rate over time and high cell viability. We observed diffuse expression of neuronal markers RNA binding fox-1 homolog 3 (RBFOX3) and neurofilament light chain (NFL), with the latter showing an increasing trend of axonal sprouting around the slices over time, attesting to their healthy condition. Moreover, we successfully transplanted into the SC-slices GFP-expressing human SC-derived neuroepithelial stem (h-SC-NES) cells at the first stages of neuronal differentiation. The NSC graft was maintained for 30 days after transplant and cells showed GFP expression for all the period in culture. The apoptotic rate of cells at day post-transplant (DPT) 30 was also found to be in line with respect to the apoptotic rate value observed at DPT 7 in the same cells40. Cells seemed to engraft into the tissue environment and survived up to several weeks.
In summary, our data demonstrate that it is possible to maintain in culture SC organotypic slices for 3 months without compromising their original cytoarchitecture and the tissue environment. Most importantly, they can be exploited to test cell therapies before proceeding with an in vivo experiment, thus reducing the costs and the experimental time. Here, we illustrate in detail all the passages to generate mouse SC organotypic slices and how to maintain them for long-term periods (&#8805;30 days). Moreover, we deeply explain how to perform NPC transplantation into the slices and how to maintain them for downstream analysis.

Autor im Rampenlicht: Langfristige Kultur von Rückenmarksschnitten zur Weiterentwicklung von Regenerationstherapien des Rückenmarks

Organotypische Schnittkultur des Rückenmarks der Maus als Plattform zur Validierung der Zelltransplantation bei Rückenmarksverletzungen

We are interested in developing a promising regenerative approach to address spinal cord injuries. In this paper, we validate spinal cord organotypic model for testing cellular replacement therapies in spinal cord research. So far, spinal cord organotypic models are maintained in culture for two or three weeks in vitro.And subculture media are suboptimal for neural stem cell engraftment, differentiation, and maturation. Cell replacement therapies still require the improvement to announce the ability of the grafted cells to reconstitute the lost circuits. Through this protocol, we provide a novel, long-term ex vivo platform to tackle cell transplant related issue like survival, integration, and maturation rate of the engrafted neural stem cells.This platform will be helpful for researchers to find the best strategy for cell transplantation, reducing the number of animals required for in vivo validation. Our protocol is simple, fast, and cost-effective to perform proof of concept and optimization studies.

Immunfluoreszenzfärbung von organotypischen Schnitten mit Stammzelltransplantaten für die konfokale Mikroskopie

Zu Beginn erhalten Sie organotypische Schnitte der Maus, die mit menschlichen neuroepithelialen Stammzellen (HSCNES-Zellen) aus dem menschlichen Rückenmark transplantiert wurden. Entfernen Sie das Medium von der Unterseite der Einlegemembran und waschen Sie die Scheiben dreimal mit vorgewärmtem PBS oder DPBS von Dulbecco. Saugen Sie anschließend das DPBS an und ersetzen Sie es durch 1,5 Milliliter vorgewärmtes 4%iges Formaldehyd.Geben Sie dann einen Milliliter 4%iges Formaldehyd auf die Oberseite der Membran. Nach dreimaligem Waschen mit DPBS mit einem chirurgischen Messer die Einlegemembran umlaufend durchtrennen und mit den Scheiben von der Kunststoffkomponente der Einsätze trennen. Nach der Immunfluoreszenzfärbung tragen Sie 200 Mikroliter Eindecklösung auf einen sauberen Objektträger auf.Heben Sie die schwimmende Membran mit einer geraden Pinzette vorsichtig an und übertragen Sie sie von der 35-Millimeter-Schüssel auf ein Deckglas. Übertragen Sie anschließend die Membran auf den vorbereiteten Objektträger. Decken Sie die Membran auf dem Objektträger mit einem neuen Deckglas ab, das 100 Mikroliter Montagelösung enthält.Lassen Sie es über Nacht unter einer dunklen Chemikalienhaube trocknen, bevor Sie mit der konfokalen Bildgebung fortfahren. Die transplantierten HSCNES-Zellen im Rückenmarksgewebe zeigten eine stabile GFP-Expression über 30 Tage, was auf ein gutes Überleben hindeutet.

immunofluorescence-staining-organotypic-slices-with-stem-cell

Research

JoVE Journal

Neuroscience

Immunofluorescence Staining of Organotypic Slices with Stem Cell Transplants for Confocal Microscopy

320 Aufrufe.  University of Pisa. Zu Beginn erhalten Sie organotypische Schnitte der Maus, die mit menschlichen neuroepithelialen Stammzellen (HSCNES-Zellen) aus dem menschlichen Rückenmark transplantiert wurden. Entfernen Sie das Medium von der Unterseite der Einlegemembran und waschen Sie die Scheiben dreimal mit vorgewärmtem PBS oder DPBS von Dulbecco. Saugen Sie anschließend das DPBS an und ersetzen Sie es durch 1,5 Milliliter vorgewärmtes 4%iges Formaldehyd.Geben Sie dann einen Milliliter 4%iges Formaldehyd au...

Serie: Immunfluoreszenzfärbung von organotypischen Schnitten mit Stammzelltransplantaten für die konfokale Mikroskopie

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs.
Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (&#8805;30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

In this paper, we provide a reproducible method to generate and maintain long-term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies.

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches ...