M&m’s: multiphoton microscopy to study stem cell therapeutics in kidney organoids

Description

du sujet :

Nous avons précédemment mis en évidence (Burdeyron et al, ) la faisabilité et l'efficacité à court terme d'une thérapie cellulaire basée sur des vésicules extracellulaires (EVs) issues de cellules progénitrices urinaires (UPCs) en préservation rénale. Une limite de notre stratégie réside dans la méconnaissance du mécanisme médiant les effets bénéfiques des EVs sur le greffon.

Afin de faciliter le développement de notre stratégie vers la clinique et d'étoffer notre expertise technologique et scientifique, ce projet a pour ambition de développer une approche de suivi d'organoïdes rénaux humains sous flux par microscopie multiphoton longitudinale et dynamique.

Méthodologie et mise en œuvre :

1. Développer un système de suivi longitudinal et dynamique d'organoïdes rénaux humains sous flux par microscopie multiphoton. Les organoïdes rénaux dérivés d'iPSC seront placés dans des conditions d'hypoxie réoxygénation (HR) mimant la préservation du greffon rénal et notamment les l’IR. L'aspect microfluidique sera réalisé avec l'UMR U (Limoges)
2. Appliquer ce système à des organoïdes rénaux issus d'iPSC génétiquement modifiées pour exprimer des protéines fluorescentes spécifiques des différentes parties du néphron, permettant de décrire l'implication des différents compartiments cellulaires dans la réponse rénale à l'HR)
3. Valider ce modèle en détaillant le mécanisme d'action des EVs : nous suivrons le devenir des organoïdes soumis au stress hypoxique et traité par les EVs (types cellulaires impactés, survie, métabolisme, stress oxydatif etc.) grâce aux technologies développées précédemment.
4. Développer une pipeline d'analyse d'image afin de quantifier, mesurer et analyser les données obtenues, en transformant des données d'imagerie en mesures quantitatives pour extraire la dynamique de paramètres d'intérêt (en collaboration avec le laboratoire XLIM (UMR U, Poitiers).

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Abstract:

Kidney transplantation remains the gold standard treatment for renal failure, but faces a major organ shortage, forcing clinicians to use "extended criteria" grafts, more susceptible to ischemia-reperfusion (IR) injury. Dynamic preservation using perfusion machines allows control of IR, and we have demonstrated the efficacy of a cellular therapy derived from urine progenitor cells. Despite the protective and promising effects of this therapy, its mechanism of action remains unknown.

To elucidate this mechanism, we propose to develop a longitudinal and dynamic monitoring system for human kidney organoids under flow using multiphoton microscopy. These organoids will then be exposed to a hypoxia-reoxygenation (HR) sequence, simulating kidney graft preservation, and treated with the cellular therapy. This model will help us identify the target cells of the therapy (tubule vs. glomerulus, etc.) and potential regeneration niches, as well as metabolic modulations, oxidative stress, and other parameters that are difficult to assess in simpler (2D culture) or more complex (animal) models. The imaging data will finally be transformed into quantitative measurements to be analyzed and modeled to understand these processes.

This unique approach, combining a complex in vitro human model with a dynamic, longitudinal, and non-destructive multiphoton imaging system, will help elucidate the protective mechanism and improve this promising therapy, offering better outcomes for transplant patients.

Background and rationale:

Kidney transplantation is the treatment of choice for end-stage renal disease, but its use is still limited by the availability of grafts. In France only, 11, patients were still waiting for a kidney in : for each available graft, 2.8 recipients are registered on the active waiting list ( report from the French Biomedicine Agency), despite donations from living donors. To address this shortage, "extended criteria" grafts from older or comorbid donors are used, but these are more susceptible to ischemia-reperfusion (IR) injury, an inherent step of the transplantation procedure. To control IR injury, dynamic preservation approaches using perfusion machines are employed, coupled with the optimization of preservation solutions.

By combining a complex in vitro human model with a dynamic, longitudinal, and non-destructive multiphoton imaging system, we aim to advance our understanding of the mechanism and refine the cellular therapy product. The integration of these models, expertise, and techniques presents a unique opportunity to develop an innovative, high-value system for the scientific community, while helping to rebalance the supply and demand in kidney transplantation.

Project description :

In our previous work (Burdeyron et al, ), we demonstrated the feasibility and short-term efficacy of a cellular therapy based on extracellular vesicles (EVs) derived from urine progenitor cells (UPCs) in kidney preservation. However, this approch is limity by the lack of understanding of the mechanism mediating the beneficial effects of EVs on the graft.

To facilitate the clinical advancement of our strategy and to expand our technological and scientific knowledge, this project aims to develop an approach for monitoring human kidney organoids under flow using longitudinal and dynamic multiphoton microscopy.

Experimental approach:

5. Develop a longitudinal and dynamic monitoring system for human kidney organoids under flow using multiphoton microscopy. Kidney organoids derived from iPSCs will be subjected to hypoxiareoxygenation (HR) conditions, mimicking kidney graft preservation and IR. The microfluidic aspect will be implemented in collaboration with UMR U (Limoges).
6. Apply this system to kidney organoids derived from genetically modified iPSCs. These organoids will express fluorescent proteins specific to different parts of the nephron, enabling the description of how various cellular compartments respond to HR.
7. Validate the model by elucidating the mechanism of action of extracellular vesicles (EVs). We will track the fate of organoids exposed to hypoxic stress and treated with EVs (affected cell types, survival, metabolism, oxidative stress, etc.) using the previously developed technologies.
8. Develop an image analysis pipeline. This pipeline will quantify, measure, and analyze the obtained data, transforming imaging data into quantitative metrics to extract the dynamics of parameters of interest (in collaboration with XLIM laboratory, UMR U, Poitiers).

Starting date

-10-01

Funding category

Public funding alone (i.e. government, region, European, international organization research grant)

Funding further details