Advancing Parkinson’s disease research by organoid modeling and 3D imaging [WEBINAR]

Summary

This webinar delves into the advancements in Parkinson's disease research using organoid modeling and 3D imaging technologies. Hosts from Miltenyi Biotec, inclusive of experts like Anisha Muos Buons and Christian Schwamborn, expounded on using PSC-derived mid-brain striatal assembloids and 3D light sheet microscopy to better understand Parkinson's pathology and accelerate therapeutic discovery. The presentation highlighted Miltenyi’s innovative solutions enabling researchers to analyze cellular interactions intricately within organoids. Key discussions also covered ethical considerations, personalized organoid models, and imaging methodologies suitable for in-depth neuron architecture studies.

Highlights

• The webinar focused on using organoid models to better understand and find solutions for Parkinson's disease. 🧠
• 3D imaging technologies, like the light sheet microscopy, provide detailed analyses of neuron networks. 🔍
• Ethical considerations in using human-derived tissues were discussed, ensuring responsible and ethical research practices. 🤝
• Miltenyi Biotec's products and workflows support a range of research applications, from reprogramming to organoid analysis. 🏢
• The potential for organoids to mimic specific brain areas was highlighted, emphasizing their role in disease modeling. 🧬

Key Takeaways

• Organoid modeling is revolutionizing Parkinson's disease research, making it more precise and personalized. 🧠
• 3D imaging techniques offer in-depth insights into neuron structures, enhancing our understanding of brain disorders. 🔬
• Ethical considerations are crucial as organoid models become more advanced and human-like. 🤔
• Miltenyi Biotec offers comprehensive workflows for stem cell research, ensuring high cell viability and efficient analysis. 🔄
• Organoids can mimic specific brain regions, offering a sophisticated platform for studying neurodegenerative diseases. 🧬

Overview

In this enlightening webinar, experts explored the use of organoids derived from pluripotent stem cells to model Parkinson's disease. The discussions centered around creating mid-brain striatal assembloids and the utilization of 3D light sheet microscopy, which collectively aid in unraveling complex neuronal structures associated with neurodegenerative disorders like Parkinson's. Such innovative methodologies pave the way for more precise disease modeling and therapeutic interventions.

A highlight of the session was the in-depth discussion on Miltenyi Biotec’s workflows. From reprogramming human fibroblasts to maintaining stem cell pluri-potency, the company provides comprehensive kits that ensure efficient research processes. This meticulous approach supports researchers in understanding the intricacies of brain organoids, ultimately contributing to groundbreaking studies in neurodegeneration.

The ethical implications of advancing organoid technologies were also addressed, emphasizing the need for stringent regulations and ethical oversight as these models become increasingly sophisticated. The discussion fostered an appreciation for the delicate balance between innovation and ethical responsibility, ensuring these powerful tools are used responsibly in the quest to combat Parkinson's disease.

Chapters

• 00:00 - 00:30: Welcome and Introduction In 'Welcome and Introduction', Iris Kabaski opens the webinar as the moderator. She introduces the main topic of discussion, which involves using PSC-derived mid-brain striatal assembloids and a 3D light sheet microscopy workflow. The discussion will feature speakers Anisha Muos Bu Yans, Christian Schwamborn, and Juan Eduardo Rodriguez Gatika.
• 00:30 - 01:00: Speaker Introductions and Webinar Sponsor The chapter begins by expressing gratitude to the webinar's sponsor, Mileni Biotech, highlighting their support for advancing research on Parkinson's disease and therapeutic discoveries. Following the acknowledgment, the speaker introduces Anisha Muos Buon, who holds a doctoral degree in biology from the University of Cologne with a specialization in neurodegenerative disorders. Post her studies, she joined M Biotech and is currently serving in a global capacity for the organization.
• 01:00 - 01:30: Webinar Agenda and M Biotech Overview The chapter introduces key figures: the product manager for the Neuroscience portfolio, Anisha, and Christian Schwamborn, the head of the Developmental and Cellular Biology group at the Luxembourg Center for Systems Biomedicine. The chapter focuses on their roles and contributions to innovative neuroscience research solutions, from basic to translational stages. It also mentions Schwamborn's position as a professor at the University of Luxembourg and his background in biology, highlighted by his PhD.
• 01:30 - 02:00: Workflows for PSC-Derived Organoids Workflows for PSC-Derived Organoids: This chapter likely discusses the various methodologies and protocols for developing organoids from pluripotent stem cells (PSCs). It highlights the career of a scientist associated with these developments, noting his experience at the University of Monster, his post-doctoral research at the Institute for Molecular Biotechnology, and his roles in co-founding innovative biotech companies like Brain Engineering Technologies and Organo. These experiences suggest a strong background in both academic and commercial applications of PSC-derived organoid technology.
• 02:00 - 02:30: Sample Preparation for Light Sheet Microscopy This chapter focuses on the preparation techniques for light sheet microscopy, particularly emphasizing potent stem cells used in the development of brain organoids. It also introduces Juan Eduardo Rodriguez Gatika, a notable post-doctoral researcher involved in experimental epileptology and cognition research. The chapter highlights his educational background and contributions to the field.
• 02:30 - 03:00: Imaging Techniques and Ultra Microscope Blaze The chapter titled 'Imaging Techniques and Ultra Microscope Blaze' discusses advanced imaging techniques used in neural connectivity research. The focus is on circuit analysis of neural connectivity using 3D image data sets. These data sets are acquired through a combination of expansion microscopy and light sheet fluorescence microscopy. The session begins with an introduction from a researcher from the University of Bonn, who thanks the audience for attending the webinar and shares a disclaimer before proceeding with the detailed discussion.
• 03:00 - 03:30: Introduction to Parkinson's Disease and Its Challenges The chapter provides an introduction to Parkinson's Disease (PD) and explores the challenges involved in understanding and modeling the disease. It begins with an outline of the session's agenda, highlighting the emphasis on integrated workflows for Parkinson's Disease using cerebral organoids. The session is set to feature Professor VOR, who will discuss the application of pscd meat brain organoids and asids in modeling Parkinson's Disease. Following this, Dr. Rodriguez GAA will delve into the concept of light sheet microscopy and will explain the use of Ultra Microscope Blaze for imaging organoids and assemblages, offering insights into recent advancements in imaging techniques relevant to PD research.
• 03:30 - 04:00: Modeling Parkinson's Using Midbrain Organoids The chapter titled 'Modeling Parkinson's Using Midbrain Organoids' begins with a brief introduction to the foundation of Milon Biotech, a biotechnology company established 35 years ago in Germany. The company currently employs over 4,900 people, with more than 20% dedicated to research and development, emphasizing its commitment to innovation in providing solutions for researchers and clinicians. Although the provided transcript is incomplete, it hints at a Q&A session that aims to address various questions related to the topic of modeling Parkinson's disease using midbrain organoids.
• 04:00 - 04:30: Exploring Parkinson's Disease Hallmarks In this chapter, the focus is on the exploration of Parkinson's Disease hallmarks. The discussion begins with an overview of the tools and products available. At Milon Iotech, they provide integrated workflows supporting various applications, emphasizing their role in PSCd organoid research. The chapter introduces a reprogramming workflow which utilizes the Stem Max IPS mRNA reprogramming kit, designed for efficient and rapid conversion of human fibroblasts into IPSCs.
• 04:30 - 05:00: Therapeutic Testing with Midbrain Organoids The chapter titled 'Therapeutic Testing with Midbrain Organoids' explores methods for maintaining and cryopreserving pluripotent stem cells (PSC) using PSC Brew media. This media is noted for its flexibility in feeding schedules, ensuring that the potency of cells is not compromised. The chapter also highlights a comprehensive cell culture portfolio that allows for flexible selection of solutions tailored to PSC differentiation needs. Additionally, it briefly touches on the workflow for cellular analysis, which begins with the preparation of organoids for analysis.
• 05:00 - 05:30: Developing Midbrain-Striatum Assembloids The chapter 'Developing Midbrain-Striatum Assembloids' discusses the method for gently dissociating organoids. It combines mechanical dissociation with gentle Max dissociators and enzymatic dissociation using a neural tissue dissociation kit, ensuring maximal cell viability. The process results in a single cell suspension suitable for downstream applications, including flow cytometry, genomic analysis, or cell culture. It also touches upon its application in 3D imaging workflows.
• 05:30 - 06:00: Functional Synapses in Assembloids The chapter titled 'Functional Synapses in Assembloids' explores the use of advanced microscopy techniques to study the complex neural networks within organoids. The focus is on combining workflows for a detailed understanding of organoid models, analyzing from single cellular components to entire neural architectures and interactions. This webinar hones in on the application of 3D imaging workflow analysis, emphasizing its role in visualizing and understanding neural interactions and architectures within these models.
• 06:00 - 06:30: Inducing Aging in Organoid Cultures In the chapter titled 'Inducing Aging in Organoid Cultures,' the text discusses the procedures for visualizing neuron architecture within organoids, specifically using light-sheet microscopy. The initial step highlighted is the staining of organoids, which is crucial yet often challenging as it requires significant time and resources to optimize. However, the text provides a solution by introducing validated antibodies optimized for 3D moof fluorescence, which are designed to improve deep tissue penetration, thereby facilitating more efficient and effective staining processes.
• 06:30 - 07:00: Concluding Remarks on Parkinson's Models The chapter discusses the benefits of using specific antibodies in Parkinson's models. These antibodies are conjugated to fluorochrome dyes, eliminating the need for secondary antibody incubation and thereby providing faster results. Additionally, the antibodies are recombinant, allowing for the generation of reproducible data. The chapter also includes a comparison of two beta3 tubulin antibodies, highlighting their differences.
• 07:00 - 07:30: Introduction to 3D Light Sheet Microscopy The chapter introduces 3D Light Sheet Microscopy, emphasizing the importance of using validated antibodies for accurate visualization of structures within organoids. It highlights the role of clearing steps in successful visualization and encourages exploring their website for suitable 3D immunofluorescence validated antibodies for various experimental needs.
• 07:30 - 08:00: Combining Light Sheet with Expansion Microscopy This chapter discusses the integration of light sheet microscopy with expansion microscopy techniques. It introduces the Max clearing kit, an organic solvent-based solution known for its fast and efficient clearing results. Importantly, the kit is non-toxic, providing a safer alternative to other methods. The chapter also highlights optimized clearing protocols tailored for different tissue types, available online. Further, it presents an example, detailing the steps and timing involved in preparing an organoid for 3D imaging using these reagents.
• 08:00 - 08:30: Pipeline for Imaging Organoid Architecture The chapter titled 'Pipeline for Imaging Organoid Architecture' discusses the final step of using the ultra microscope Blaze, a fully automated microscope. This device allows users to image samples automatically, enabling the performance of other experiments simultaneously. The microscope is noted for its user-friendly features and advanced optics, which facilitate high-resolution imaging from whole mouse specimens to small organoids. The chapter highlights a video featuring a recent release intended for high-throughput 3D imaging, demonstrating the capabilities of the Ultram mount 48.
• 08:30 - 09:00: Analyzing Mature Neurons in Organoids In this chapter titled 'Analyzing Mature Neurons in Organoids,' the focus is on the experimental process of imaging and analyzing cerebral organoids. The R&D team successfully generates cerebral organoids, differentiating them over a 30-day period. These organoids undergo staining with several markers: the proliferation marker k67, the neuronal marker beta3 tubulin, and the neural stem cell marker Spa 6 and so 2. Additionally, all organoids are cleared using the Max clearing link method, facilitating comprehensive imaging analysis. The imaging process allows for the simultaneous analysis of up to 48 organoids in one run.
• 09:00 - 09:30: Case Study: Assembloids for Neostriatal Pathway In this chapter, the focus is on a case study of assembloids for the neostriatal pathway. The speaker, Professor Yan Schwamborn from the University of Luxembourg, discusses modeling Parkinson’s disease using patient-specific midbrain organoids.
• 09:30 - 10:00: Summary and Group Acknowledgments The chapter begins with disclosures regarding the licensing of certain technologies and molecules to a company called Organ Therapeutics, which was co-founded by the presenter. The main topic discussed is Parkinson's disease, highlighting it as an escalating issue primarily due to the aging populations in societies across the western and other parts of the world.
• 10:00 - 10:30: Question and Answer Session The chapter titled 'Question and Answer Session' begins with a discussion on the increasing prevalence of Parkinson's disease, which is associated with aging. It references a curve from a paper published a few years ago, indicating that the current estimates of Parkinson's patients have exceeded previous projections, with an estimated 10 million patients worldwide. The chapter emphasizes the growing societal burden of Parkinson's as the numbers continue to rise. Furthermore, it highlights that Parkinson's is a neurodegenerative disorder that is likely familiar to the audience.
• 10:30 - 11:00: Ethical Implications of Organoid Research The chapter discusses the ethical implications of organoid research, focusing on the diverse symptoms patients experience due to certain diseases. It distinguishes between motor symptoms like rigidity and tremors, and non-motor symptoms such as sleep disturbances, anxiety, and hyposmia. The histopathological aspects of conditions affecting dopaminergic neurons, which are responsible for producing neurotransmitter dopamine, are also highlighted. This sets the stage for a deeper exploration of organoid research's ethical considerations.
• 11:00 - 11:30: Digestion Process for Sample Transparency The chapter 'Digestion Process for Sample Transparency' explains the role of dopaminergic neurons located in the substantia nigra of the midbrain. These neurons project to the stratum and are responsible for secreting dopamine, which is crucial for muscle movement control. In certain disease conditions, these neurons degenerate, leading to lower dopamine levels and resulting in movement-related symptoms. The chapter also discusses the characteristic dark color of the substantia nigra, which is due to neuromelanin.
• 11:30 - 12:00: Applications of 3D Imaging 3D imaging can reveal differences in neurological conditions, such as Parkinson's disease, where dark staining neurons disappear and protein aggregates emerge. These aggregates, called Lewy bodies and neurites, are intracellular and linked to disease progression. Parkinson's disease shows both risk factors and inverse risk factors.
• 12:00 - 12:30: Integration with Patient-Specific Models This chapter discusses the integration of patient-specific models in understanding the risk factors for Parkinson's disease. It covers the genetic components, indicating that around 10% of cases are monogenetic while others are idiopathic, involving complex genetic or environmental elements. Aging is highlighted as a major risk factor, and environmental factors such as pesticides and air pollution are known to significantly contribute to the risk of developing Parkinson's disease.
• 12:30 - 13:00: Technical Aspects of Light Sheet Imaging The chapter "Technical Aspects of Light Sheet Imaging" discusses various risk factors for diseases, including inverse risk factors like female gender and lifestyle choices such as a healthy diet and exercise. Interestingly, it mentions caffeine consumption and smoking as unexpected inverse risk factors. Additionally, genetic factors play a protective role. To study Parkinson's disease, the chapter highlights the use of human midbrain organoids in laboratory settings.
• 13:00 - 13:30: Concluding Remarks and Contacts This chapter discusses the initial steps of a research process that begins with patient samples, such as skin biopsies, blood, or urine-derived cells. These samples undergo a reprogramming process using Yamanaka factors to become induced pluripotent stem cells, which are similar to embryonic stem cells. Further differentiation into neuroepithelial stem cells with midbrain and hindbrain identity is described, indicating the beginning of a complex scientific procedure.

Advancing Parkinson’s disease research by organoid modeling and 3D imaging [WEBINAR] Transcription

• 00:00 - 00:30 [Music] hello everyone and welcome to today's webinar I'm Iris kabaski associate science editor for the scientist and I will be moderating our discussion today Anisha muos bu yans Christian schwamborn and Juan Eduardo Rodriguez gatika will discuss using PSC derived mid-brain striatal assembl oids and a 3D light sheet microscopy workflow highlighting
• 00:30 - 01:00 value not only to elucidate Parkinson's disease related pathome mechanisms but also to accelerate the discovery of potential Therapeutics before we begin I'd like to thank mileni biotech for sponsoring today's webinar and with that let me introduce our speakers Anisha muos Buon received her doctoral degree in biology from the University of Cologne Germany in 2023 with a focus on neurodegenerative disorders after her studies she joined M biotech and currently is the global
• 01:00 - 01:30 product manager for the Neuroscience portfolio closely working together with research and development Anisha focuses on innovative solutions that support Neuroscience research from basic to translational stages yence Christian schwamborn is the head of the Developmental and cellular biology group at the Luxembourg Center for systems biom medicine as well as a professor at the University of Luxembourg he obtained his PhD biology
• 01:30 - 02:00 from the University of monster and subsequently worked as a post-doctoral researcher at The Institute for molecular biotechnology in VI he was previously the co-founder and chief scientific officer of brain engineering Technologies in 2019 and he co-founded Organo theapocryphaltruth
• 02:00 - 02:30 potent stem cells for the development of brain organoid and asmid models which are used for invitro disease modeling Juan Eduardo Rodriguez gatika is a post-doctoral researcher in the functional neuroc conomics group of Martin Schwarz at The Institute of experimental epileptology and cognition research of the University of bond Medical Center completed his PhD in the biophysical chemistry group led by olich
• 02:30 - 03:00 kek at the University of Bon his current research involves circuit analysis of neural connectivity in 3D image data sets acquired by combining expansion and light sheet fluoresence microscopy okay let's make sure our slides are up and runting thanks Diana for your kind introduction first of all I want to send a warm welcome to all of you thank you for joining us at today's webinar here is my disclaimer before we start I will
• 03:00 - 03:30 present you today's agenda I will kick off the session with a brief introduction into the integrated workflows for pscd cerebral organoids that we offer at milon biotech then I will hand it over to our first Speaker Professor VOR who will talk about the use of pscd meat brain organoids and asids to model Parkinson's disease afterwards Dr Rodriguez GAA will explain what is light sheet microscopy and how can we use the ultra microscope Blaze to image organoids and assembl
• 03:30 - 04:00 oids finally we will open the floor for a Q&A session to address the questions you may have milon biotech was founded 35 years ago in Germany currently we are more than 4,900 employees with more than 20% of us dedicated to research and development this fact highlights our strong commitment to develop innovative solutions to researchers and clinicians with more than 19,000 and
• 04:00 - 04:30 products comprising instruments reagents and consumables we are able to offer integrated workflows for a vast range of applications at milon iotech we offer integrated workflows that support your pscd organoid research first we have a reprogramming workflow that uses our stem Max IPS mRNA reprogramming kit which has been designed for rapid and highly efficient reprogramming of human fiber blast into IPS
• 04:30 - 05:00 next our PSC maintenance and cry preservation workflow with our PSC Brew media is designed to allow flexible feeding sheds without compromising pry potency of the [Music] cells moreover our broad cell culture portfolio allows you to flexibly select the perfect solutions for your PSC differentiation needs once you have your organoid or asmid to analyze them in one hand the cellular analysis workflow starts with
• 05:00 - 05:30 the gentle dissociation of the organoid to do so we combine the mechanical dissociation with our gentle Max dissociators together with enzimatic dissociation provided by the neural tissue dissociation kit this mechanical and enatic dissociation combination ensures maximal cell viability the resultant single cell suspension is ready for DST string applications such as flow cytometry genomic analysis or cell culture on the other hand the 3D imaging workflow use
• 05:30 - 06:00 slid sheet microscopy to allow you to study the complex neural networks within your organoid the combination of these two both uh two workflows allows you to gain an in-depth understanding of the organoid model from the analysis of single cellular components to the visualization of the neural architecture and the neural interactions but for today's webinar we will focus on the application of the 3D imaging workflow analysis is of
• 06:00 - 06:30 organoids and asids to visualize the neuron architecture of the organoid we need to follow a series of steps to prepare the sample for the light sheet microscopy the first step is the staining of the organoid we know that optimizing a staining takes a lot of time and resources and not always means you will have good results to make it easier for you we have validated our antibodies for 3D moof fluoresence and optimize them for deep tissue penetration this will help you reduce
• 06:30 - 07:00 the time and cost to invest during the optimization phase in addition our antibodies are conjugated to our fluorochrome dice which will lead you to obtain faster results since you don't need to incubate with a secondary antibody also our antibodies are recombinant which will enable you to generate reproducible data here at the right you can appreciate the differences between two beta3 tuing antibodies at the left is sustaining
• 07:00 - 07:30 with a nonv validated nonoptimized antibody and at the right is our beta 3 tuing antibody as you can appreciate already validated antibodies will allow you to visualize the different structures within the organoid you can brow through our website to find out more 3D imunofluorescence validated antibodies that can fit your experimental needs the clearing step is crucial if you want to visualize ize the 3DS
• 07:30 - 08:00 structure using light sheet microscopy our Max clearing kit is organic solvent based and provides you with fast and efficient clearing results it is non-toxic which makes it safer than other clearing methods in addition we have optimized clearing protocols for different tissues that you can find online on our website here you have an example of the steps and timing for the preparation of an organoid for 3D imaging using our reagents
• 08:00 - 08:30 the last step of the workflow is the Imaging using the ultra microscope Blaze which is a fully automated microscope meaning the microscope will image your samples automatically but you can do other experiments in the meantime the microscope is easy to handle and has Cutting Edge Optics that allow you to obtain high resolution images from Whole Mouse to small organoids in this video you can see one of our L last releases for high throw with 3D imaging the Ultram mount 48
• 08:30 - 09:00 which allows you to place up to 48 organoids and image them in one run for this video our R&D team generated cerebral organoids and differentiated them for 30 days the organoids have been stained with a proliferation marker k67 the neuronal marker beta3 tubulin and a neural stem cell marker Spa 6 and so 2 in addition all the organoids have been clear with our Max clearing link it
• 09:00 - 09:30 following the protocols we have available in our website and with this I will now hand it over to our first invited speaker Professor shambor from the University of Luxembourg hello my name is Yan schwamborn and I'm going to talk about modeling Parkinson's disease in patient specific midbrain organoids and actually also brain stum
• 09:30 - 10:00 assembl before going into the actual presentation here is a disclosure some of the uh Technologies of the um approaches of the molecules that I'm going to present in this presentation are licensed to the company organ Therapeutics that I co-founded a while ago so Parkinson's disease is um an increasing problem for our societies because particularly in the western world but also in other parts of the world societies are significant aging so
• 10:00 - 10:30 we are all becoming older and older and Parkinson is age Associated you just see here a curve from a paper that has been published already a couple of years ago and actually the current numbers are already higher than what is projected here we're currently estimate probably having about 10 million parkone patients and numbers are strongly increasing so it become a higher burden for society what is Parkinson Parkinson is a neurod degenerative disorder and um I think all of you already have seen
• 10:30 - 11:00 patients with this disease particular um identified on the motor symptoms these are Trea banesia rigidity many others but there's also the non-motor symptoms that are pretty diverse and different patients suffer from different of those non-motor symptoms they include sleep dis disturbances hyposmia anxiety and many other aspects on the histopathological levels in um healthy conditions dopaminergic neurons so neurons produce the neurotransmitter damine have their cell
• 11:00 - 11:30 bodies in the substantia of the midbrain and there they project to the stratum where they then secrete dopamine and that is important particular for muscle movement control while in the disease situation these dopaminergic neurons degenerate as a consequence levels of dopamine in thisum are much lower and it comes to the symptoms um that I've just mentioned before and since the substantial um has its color its dark color from neur melanine
• 11:30 - 12:00 we can see that under normal conditions this dark staining is present but it's absent in the Parkinson conditions just because these neurons are degenerating on the other s on the other hand um the disease goes hand in hand with the appearance of protein aggregates consisting of the protein Alpha cocine and this is called louisology that can be Lou bodies being intracellular actually in the neurons or in the neurites and then are called L neurites Parkinson's disease has risk factors as well as inverse risk factors
• 12:00 - 12:30 risk factors are genetic factors there about 10% of all cases are clearly monogenetic but there's also a larger population of idopathic which can have a complex genetic have um environmental contributions or just an unknown cause aging is probably the major risk factor so the older somebody gets the more likely it is to suffer from Parkinson's and I already mentioned that environmental factors particular uh for example pesticides but also air pollution strongly contribute to the risk to suffer from Parkinson's there's
• 12:30 - 13:00 also inverse risk factors including female gender um certain lifestyle factors obviously a healthy lifestyle with uh Sports uh com complete diet are helpful but also unexpected things like coffeine consume and uh smoking seem to be inverse risk factors and then there's genetic factors that are also protective so our approach to model Parkinson's disease in the lab is the usage of uh human midbrain organoids so
• 13:00 - 13:30 everything that we are doing is starting with patient samples typically it's can be skin biopsies but it could be also for example blood or even cells uh that you can find in urine so these cells are then reprogrammed with the yamanaka factors into induced plent stem cells these are stem cells that are very similar in all their characteristics to embryonic stem cells we further differentiate them into new epical stem cells that are already prepatterned for midbrain hindbrain identity and then we differentiate Z into the actual midp
• 13:30 - 14:00 organoids and the reasons for doing this intermediate step is by restricting the differentiation potential the midp organoids that we get out of this process are actually highly reproducible just in a nutshell what are midbrain organoids these are 2 to 4 mm size three-dimensional structures have midbrain identity so they express transcription factors like Foxy 2 or lmx1 that um identify as these as as midbrain cells they have high levels of damic urans you see here the th staining for dopaminergic neurons but you also
• 14:00 - 14:30 can see dopamine in green here um itself being produced within those cells the rbin organoids produce neom melanine which is quite interesting because this is a feature that you would for example not find in rodents and that's actually a feature of maturation already indicating us that although these organoids are probably young because they are derived from embryonic cells they do have features of maturity mid organes show a tissue organization so we have a layering of cells and we not only have neurons but
• 14:30 - 15:00 we also have Gia cells and other cell types and the neurons in there show elector physiological activity a subset of those neurons actually also show pacemaking activity firing with a frequency of about six Herz we used uh singles and RNA sequencing approach to get a better understanding of uh the actual cellular composition of those midbrain organoids and that was done together with people uh in Luxembourg like Alex scupin lab as well as the Rican Institute in yok arm and what we can see is if we compare
• 15:00 - 15:30 midp organoid of a day of 35 and 70 days is that the cellular complexity is increasing so we get um more cell types when we continue to differentiate them and we not only find damic neurons but also other neural cell types just as an example we do see for example glutamatergic gabaergic or serotonergic neurons so we have really a huge degree of complexity
• 15:30 - 16:00 now having these mpin organoids in hand we obviously want to use them for invitro modeling of Parkinson's and um what we did in order to do that is looking for the three main features that Parkinson probably has this is a loss of dopaminergic neurons the appearance of alpha cocine Aggregates and neur inflammation so let's first look at the loss of dopaminergic neurons here we differentiated um induced pent stem cells with IM mutation in the L 2 Gene a mutation that leads topd either in 2D or
• 16:00 - 16:30 in 3D in dopaminergic neurons or midp organoids and then simply quantifi the amount of dopaminergic neurons and strikingly in a 2d culture we would not see this loss of dopaminergic neurons that we see in a patient while in the midp organoid we can very clearly see this phenotype you can see here in the red column um the PD situation has way less dopaminergic neurons than the hethy control further we do see the appearance of protein aggregates this is here a
• 16:30 - 17:00 setting where we have um healthy Midrin organoids so from an individual that does not suffer from Parkinson's disease as well as from a Parkinson patient with the triplication of the alpha cocan Gene and you can see in the healthy situation there's a bit of cocan in the green staining but we do not have the phosphor related version the p129 version of alpha cocan which is a hmark of the posology while in the disease situation we have way more Alpha the green staining is way more intense but we also particularly find these aggregated p129
• 17:00 - 17:30 positive structures and if you zoom in there in more detail you can even see that they form quite diverse morphologies pretty similar actually to um what we see in the patient brain and to highlight this in a bit more uh detail we compared the different morphologies of these Aggregates in the organoid to what we actually see in um in the patient brain and postmortem samples and again we do find that many of the structures we can see in the mid brain organoids are similar to what we
• 17:30 - 18:00 see in the patient brain convincing us that we really can hear recapitulate pesy to a good extent and importantly um in this setting here we do not trigger in any way this is really endogeneous just because the organ it comes from a patient there's no uh fiber treatment no toxin treatment it's really just endogenous and then the third characteristics is uh the recapitulation of neuroinflammation we can um look for example for the levels of uh
• 18:00 - 18:30 pro-inflammatory cyto and what we would see is that in a midin organoid for a patient with a duplication Alpha cocine we find significantly higher levels of inine 1 while in a patient with a lar 2 mutation we would find higher levels of tnf alpha it is interesting to see that in both cases we would have in general pro-inflammatory conditions but the primary cyto kindes that drive these conditions seem to be different depending on the mutation importantly we canot not only recapitulate the disease but we can also
• 18:30 - 19:00 um use strategies to develop Therapeutics and I just show you two studies here that we have published in the last years um one on the left side is with patients with a mutation in the pink one mutation where we get again mid brain organoids and you can see that in the diagram the uh patients this is these red color um have way less damic neurons than the healthy controls but then up treatment with with a drag in this case is a cyclodextrin we can rescue the phenotype to a good extent
• 19:00 - 19:30 meaning we can increase the amount of dopam neic neurons and that drug here Works in a way that autophagy is turned on while in the second study here on the right side um this is from uh induced BL poent stem cells with IM mutation in dj1 which is also Associated to Parkinson's disease which is a sply sight mutation we can Rescue by um treatment with splicing we through compounds and again you can see that the patient situation or the PD mutation situation has way
• 19:30 - 20:00 less dopaminergic neurons than the healthy control while upon treatment we can rescue the amount of dopaminergic neurons significantly so this is a model that allows us not only to describe the disease um to to a good extent recapitulate Hallmarks but also to experimentally test therapeutic approaches however you remember from the introduction um Parkinson is is a complex disorder where the doamin neic urans have their cell bodies in the midbrain and this is probably what we have recapitulated so far in the
• 20:00 - 20:30 organoids but they project their axons actually to this Trum and this is something in the model that I've shown you so far we have not recapitulated so we set out to come up with a more advanced approach where we take midbrain organoids and actually fuse them to stral organoids so organoids representing the strum and build midbrain stratum assembl loids where this projection from the midbrain to the stratum would be recapitulated
• 20:30 - 21:00 and yeah we have developed these uh mid brain stratum assembl loids and in the first attempt obviously we want to characterize them so we look for the expression of certain markers for the different uh different regions of the brain like for example as I've told you before um the midbrain would Express Fox A2 you can see that here in the red staining while the strum would Express CP 2 which is yellow staining here um we find um ascl1 again it's a straight armm we find Cory being expressed in the midbrain and obviously the really the
• 21:00 - 21:30 Cardinal markers for these regions th for the damic neurons strongly expressed in the midbrain and dar surgy to the marker of the medium spine inurance of the stratum being expressed in the stratal organoids and in this experiment as well as in many of the following we have actually labeled the Midrin organoid with gfp so we have the stem cells engineered in a way that they stably express gfp and thereby appear green we now take these um midbrain Statum assembl and subject them to um
• 21:30 - 22:00 RNA sequencing single cell RNA sequencing we not only find the various types of damic neurons for example A9 and A10 damic neurons so different regions of the midbrain but we also find a robust population of the medium spiny urans of this traum so we have the main cell types that are here relevant for us are being present because normally these dopamin orent urans would project to the stum medium spine
• 22:00 - 22:30 inurance um now to investigate that in further detail we used various Imaging and and sensing approaches that are largely done in collaboration so for example um this high resolution imaging here is done together With Ur ku's Lab at the University of Bon you again in green see here the midbrain organoid and we used then an antibody against th against the um enzyme that is expressed in damic neurons to see whether these damic neurons would actually project into thisum and you see on the upper
• 22:30 - 23:00 left an overview image but then if you zoom in and do a 3D reconstruction you can quite nicely see the dopaminergic neurons having their cell bodies in the midbrain but then sending their exons deeply into this traum and then together with Peter atas Lab at the University of Vienna um we used sensors that would be able to measure actually levels of dopamine because we not only want to see that they are connected but also that the dopamine that is produced by the damic urin in the midbrain is secreted in the
• 23:00 - 23:30 stratum and if we use these these needle sensors uh we can measure in the stratum pretty low levels of dopamine as you can see here in this figure B but if we connect this strum to a mid brain to build this assembl oid levels of dopamine in the Statum go up significantly indicating that indeed there's a functional connection with the secretion to further prove um functional synaptic connections we teamed up with Rebecca mata's Lab at The Institute
• 23:30 - 24:00 pastur in asence and they used a rabis virus approach where you do uh sequential infection with two viruses having a green and a red fluoresence and um just to explain that very briefly whenever you would see um red fluoresence in the midbrain organoid you would know that there is a functional synaptic connection between statom and midbrain because these um en this virus uh allows a retrograde labeling and it's
• 24:00 - 24:30 only transmitted in active copses and as you can see particular in the higher magnification images down here on down on the right side of the slide you see um red fluorescent cells that are even positive for the th marker and these are dopaminergic neurons that have functional connections to this traum and they have their cell bodies in the mid brain and then to really finally see that this uh functional connection also goes hand in hand with the firing of action potentials um we use microfic
• 24:30 - 25:00 devices that are developed by Regina lutkus Lab at the Technical University of einthoven where we have two compartments um on the upper upper left here we would have the midp organoid and the compartment with the midp organoid is with channels connected to another compartment where would have the stratum organoid and at the bottom of these channels we have electrodes spotted so whenever um an action potential is now fired from one side to another we would see an activation of these electrodes
• 25:00 - 25:30 because it's an electric signal at the end of the day uh and by doing that we get what you see here on the lower left side of the slide namely rata plot of um well neuronal activity together with uh Mario botti's Lab at the University of Padua and at Vim in padwa we were able to not only really measure this activity but making sense out of it in a way that we can see directionality and the Striking thing is it's nearly exclusive directionality from the midbrain to the stratum so we see lots of firing and this firing in
• 25:30 - 26:00 really the majority of the cases goes from the midbrain to the stratum now knowing that the majority of the uh neurons in the midbrain are actually dopaminergic neurons we can be pretty sure that many of these signals that we see here are actually from damic urans that uh inovat the stratum and they um activate or or or signal to medium spine inurance um finally to to conclude these present a I just want to have a glimpse
• 26:00 - 26:30 on on the induction of Aging because as I've told you um Parkinson's disease is largely driven by aging or aging is the major risk factor so uh we wanted to also mimic this aspect of the disease in the dish and for this we teamed up with Frank edenhofer Lab at University of inbrook who has a strong expertise in the induction of Aging by overexpression of pine so Pine you probably know from the disease pereria um this induces a premature aging so we have here now uh used IPS cells where we can induce the
• 26:30 - 27:00 expression of pine by uh by doxy cycling treatment and that also um leads to the expression of gfp if we do that we can on the one hand see that progressively from day 30 to day 60 the um expression of certain aging markers increases uh we here for example have looked at the H2 a x marker and we can very clearly see that this up treatment with doxy cycling increases a bit at day 30 but it particular strongly
• 27:00 - 27:30 increases at day 60 so we can progressively induce aging and we looked at many more markers but I just want to show you very quickly uh just to stay in time that we not only induce aging but that this aging also goes hand inand with the induction of cence this is a better G staining here and better G staining indicates Cent cells and you see that in the wild type situation so without Prine we hardly see that we see here and there a single cell but not massively and also in the engineered
• 27:30 - 28:00 cells that are untreated so where the Prine is not expressed we will not see senescent cells but in the engineered cells where we indued expression by doxy cycling treatment huge amount of sessen cell is coming up so we can experimental here quite nicely induc inessence and also in an RNA sequencing approach we very clearly see that these engineered cells expressing Pine cluster very differently different or far away from the other cell types where would not induce artificial aging we find in those
• 28:00 - 28:30 cells also hm marks of Parkinson's disease this is all summarized in the manuscript um that's currently in revision and hopefully when this webinar comes out is already public so what I've shown you is um that we can build midb organoids from Human stem cells they recapitulate many aspects of Parkinson's disease uh when they come from from patient specific cells and we can by now also build Midrin spum asids and in particular we can also induce Aging in these cultures there's many more developments in that direction so we're building more complex assembl loids
• 28:30 - 29:00 aiming at having an as good as possible representation of Parkinson's in the dish and um yeah with that I would like to thank my team at um the Luxemburg Center for systems by medicine at the University of Luxemburg um all collaborators I hope I have mentioned everybody who's involved in that study and particular obviously also the funders that made this research possible and I thank you for your attention thank you Professor isor for this
• 29:00 - 29:30 insightful presentation on modeling Parkinson disease using organoids and asmid now let's welcome our next speaker Dr Rodriguez gatia who will share his expertise on 3D light sheet microscopy and its advantages for organoid and asmid imaging today I would like to present you um this uh new technique or or or pipeline that was optimized in order to get most of the information of um 3D
• 29:30 - 30:00 samples like organoids so first our motivation is uh that well you know that depending the type of samples that you have you will use a a specific type of microscopy method in general light microscopy have the limitation of the defraction of light that is about 250 nanometers and if we want to overcome that limitation then we
• 30:00 - 30:30 have to go to the super resolution microscopy unfortunately these methods are doesn't allow to image a sample in depth that is more than 300 micrometers so what's happening if we have a sample a 3D sample like an organoid right that is between 1 mm and 1 cm in terms of size but and and we will to see cellular or subcellular
• 30:30 - 31:00 interaction let's say synapses synapses are in the range of about 100 nanometers so uh that fits with super resolution microscopy but if we want to preserve right the complete information of the sample then we cannot use that type that type of techniques so the question is how to achieve detailed Imaging of these subcellular features so first how to get sub resolution features right when we image
• 31:00 - 31:30 a point source our microscope is introducing some Distortion and that point doesn't look like a point anymore but so we have that the area pattern or the deformation that is caused caused by the by the point Sprint function and if we have two points that are too close to each other then we are not able to resolve In classical light microscopy so super resolutions try to Sharp this
• 31:30 - 32:00 psf right but they have the disadvantage that you need really specific equipment and still you will not be able to see samples that are B more than 1 mimer in size so another way is to use expansion microscopy where we physically separate this uh the the the elements in our sample so how expansion works right so let's imagine that we
• 32:00 - 32:30 have a sample like that a cell and we would like to analyze one of the features like micro tools so first what we normally do after fixation is to label that either with antibodies uh with a direct conjugation primary secondary or expressing fluorescent proteins then is when the expansion protocol start and what we uh do is to introduce a Linker molecule that is catching the amine groups of the
• 32:30 - 33:00 proteins and in the other side will get attached to the PO to a polyamid gel that we are inserting into the sample then in the next step the digestion or homogenization of the sample what we do is to try to remove everything every element that is not attached to the gel right to this to this polyamid 3D GD that we are inserting into the sample so everything that is is not attached to that it's washed away and we just have a gel with
• 33:00 - 33:30 the fluorophor or part of the proteins retain and because of this gel is uh hydrophilic if we put it in water right we have this water dialysis and the gel will start to grow in size so here's an example of a mouse R section we can see the before and after so after what do we have we have a
• 33:30 - 34:00 completely transparent sample we have a reflective index like water that means that we don't have the the needs right to use uh a special objectives we can use normal objectives and um the original protocol was made to work with samples that were about 100 micrometers uh in thickness and and had a weak preservation of flues proteins so we went through a process of
• 34:00 - 34:30 optimization in terms of permeabilization linker penetration of the elements the expansion buffer and also the digestion buffer and now we are able to preserve fluorescent proteins and we can work now with samples that are 20 times thicker than was reported in the original protocol so how to image this type of samples so well if you go to uh confocal
• 34:30 - 35:00 that is like the the standard way of Imaging for biological samples what we are doing is uh exciting the fluorescent molecules of the sample and then with the same objective we are getting back right the exitation of those molecules so if we and we have to do that point by point if we want to image a complete plane and this process is a slow right because we have to go
• 35:00 - 35:30 Point by Point through the complete image plane but it's also damaging a little bit the the sample in terms of photo bleaching or photo damage because we are always Illuminating the complete sample above and below the image plane that we are interested so one way to overcome this problem is with liting microscopy where we have the illumination and the uh adquisition in a um perpendicular right
• 35:30 - 36:00 in in in an orthogonal way so like this so we have an objective that is just dedicated to the illumination of the sample and another one right acquiring so if we generate this sheet of light right the objective can acquire the the field of view at once without any problem so it's much faster so it's we can compare that with the EP
• 36:00 - 36:30 fluoresence but with the advantage that we have really low autofocus illumination so one of the advantages of Ling microscopy is that it's really fast compared to confocal we have very little photo damage almost no autofocus excitation but we have the drawback that is really sensitive right to absorption and scattering of the light luckily this is not a problem if we work with expansion microscopy
• 36:30 - 37:00 because as I mentioned before we will have a sample that is completely transparent so in the beginning some years ago we we combined and we um developed this technique that the combination between leit and expansion that we name lit fluorescent expansion microscopy in order to take the best of both uh techniques right so because with expansion you will have the drawback of have a
• 37:00 - 37:30 large sample so if you put that under the confocal Imaging time is increase and you have and by increasing the image in time you also have more risk to photo bleach your sample so with uh lied we can go through big tissues big samples quite fast but we also have a drawback that is that we will generate a lot of data so that's why the first time with that with our
• 37:30 - 38:00 custom build setup where we have also a revolver in order to change magnifications uh we suggest that the best way is to depending what do you want to image go for a specific magnification of your sample because if we consider an expansion of four times right that that we make our sample four times bigger uh that of course will increase the resolution of our microscope by four so we could achieve
• 38:00 - 38:30 for example lateral resolutions of 90 nanometers but at the same time if want if we want to image one cubic millimeter we will be generating about 8 terabytes of data right so and that's all in one channel so that's why what we suggest is to have like a a multiscale approach with your samples now an example how we can analyze right the cellular and subcellular architecture of 3D samples
• 38:30 - 39:00 like the organoids so what do we need so we would like to preserve the 3D architecture especially because people is generating these nice 3D samples that try to recapitulate what is happening in Vivo so we don't want to have that nice 3D sample and then cut it into slices because we will generate artifacts we will lose information and we don't want uh we don't want that so we need to be able to
• 39:00 - 39:30 get a whole Imaging a whole or organoid mounting and imaging and get super resolution features so here you can see um a pipeline that we report uh in 2022 so with a fix fixed brain organoids we suggest some permeabilization but here you can use any antibod staining with commercial antibodies and then is when the the expansion this optimized expansion
• 39:30 - 40:00 protocol start uh uh start playing a role so we embed our sample in a gel with digest in order to have a transparent sample and then if you expand your sample in water you can expand you will have a sample that is four times bigger but you don't have to put it in water necessarily so you can also put it in PVS and then your sample will be only 1.5 the original size and
• 40:00 - 40:30 what is nice is that you can ex even if you have a start with your sample in PVS then you can wash it with water expand it four times and after Imaging you can wash the sample again with PVS and shrink it back so here you can see in the upper part an example of an organoid of about a bit more than 1 mm in 1 mm in diameter
• 40:30 - 41:00 right embedded in the gel so you can see the original size then is exactly the same gel expanded 1.5 and four times and the image is the um Optical section of the organoid that Weir with the lies so com with with this right um features that we can expand right the the our sample our comp complete sample uh in different uh with different expion
• 41:00 - 41:30 expansion factors plus the ability to use a li with different magnification objective we recommend to go to different scales right and for example in the mesos scale image the complete organoid if you want to have let's say the organoid volume the surface area or if you want to see in this case ret distribution in the micro scale you can see cleavage planes uh you can recognize a specific
• 41:30 - 42:00 interneurons that are usually difficult to find if you just work with the slices right because if you have um a distribution of few cells inside of the whole volume you usually have the chance that you will take a slide and you will not find nothing there and then in the nanoc scale as I will show you we can we are able to find dendritic spines and also uh to measure synaptic distances what is important right for for the
• 42:00 - 42:30 people who is working with organoids is uh to get most of the information out of that and as you can see in confocal you have the problem that when you are going deep into the sample then you are not able to get that much of the information even if the antibody penetrates there right because your sample is not transp transparent and you have a a scattering in of the light so here is down is exactly the same organoid That
• 42:30 - 43:00 Was Then mounted and and expanded and image with the light sheet and here we can see clearly right the information that was hidden uh when we use the RW sample and conf foca also structural Integrity of your sample the idea is that if we expand this sample in a isotropical way right that there are no deformation so here we can see some gfp positive
• 43:00 - 43:30 projections In The Raw sample then we found exactly the same projections after the treatment and already you can see that one of the advantage is the reduction of the backr if we compare both here you can see that the difference is really minimum or minimal and that is because the digestion was optimized right in order to um to to eliminate right everything that
• 43:30 - 44:00 is not attached to the Y but at the same time be gentle is gentle enough to preserve the fluoresent proteins because there what you can see is a expression of egfp without any antibody uh and we don't want to lose right that information after the the complete uh procedure so here it's uh we analyze on mature neur in cortical brain organoids um again here if you see the size of the
• 44:00 - 44:30 sample is more than square cm and more than 5 mimet in terms of thickness so we use the mesoscale Imaging just to have an overview and then we focus in a small region of interest in order to get an A volume here we can see now the cells and if we focus in another smaller region of interest and we go with a higher magnification we can see that neuronal projection and in the arrows we Mark
• 44:30 - 45:00 some protrusions that in order to properly identify those are uh spines and not just an artifact of the Imaging of the image uh we Analyze That in 3D so we are able right to go to that uh level of details into the sample make a segmentation and quantify the experence unfortunately this sample that was
• 45:00 - 45:30 cultivated for 5 months we couldn't find a lot of uh synaptic interactions so that's why what we did at that time was to cultivate another sample for 14 month we use pre and post synaptic antibodies against Homer one and synapsin and again we focus or we IM much a small region of Interest as you can see in the video with the highest resolution and then the idea was to recognize right the
• 45:30 - 46:00 gfp positive projections and uh use that as a mask to qu to to recognize preoper synaptic proteins and the counterpart so that like that we are able to quantify either the amount of dritic spines that we have uh in the surface of these neuronal projections but also synaptic distances that are around 150 nanometer apart and that is below right the the
• 46:00 - 46:30 fraction limit that I mentioned in the beginning of 250 so we are able or we have the capabilities to go for super resolution um now we like to show you an example a project uh of U Dr kiraki BPA from the lab of U Professor schamber where they um produce assembl in order to get a a better model uh for the for
• 46:30 - 47:00 the neostriatal pathway so they use midbrain um and Statum organoids that they uh fuse in order to recreate the this connection and these samples have the something um special is that they are embedded in a in in like a matri gel that is called gelx and again what they would like to have is the 3D preservation of the architecture
• 47:00 - 47:30 be able to image the complete sample and in the same Imaging session be able to uh choose a specific region of interest and see if there is a projections of theox hxil positive cells from the from the midbrain to the statom assembler so this is one of the samples so you can see in blue the nuclear
• 47:30 - 48:00 staining the gfp positive cell corresponding to the midbrain organoid so it's you can see that is smaller because was cultivated only 25 days uh and we also use th to see the the connections right even if the sample here looks smaller is just because in the surrounding there is this gel Trix that
• 48:00 - 48:30 was also uh preserved in the beginning but thanks to the to the lighted Imaging and this time we use the the ultra microscope blaz from milon G with the 4X magnification we are able to scan go through the sample and see right the interaction between these two organoids that are form forming now this assembl now that we know that we have the
• 48:30 - 49:00 information right of the complete sample we would like to go to a specific region and see right if uh there is interaction between these two organites so from without taking out the sample right we just change to a different scale then we focus in this um small region of Interest where um we would like to see the interaction between the midbrain organoid and the Statum
• 49:00 - 49:30 organoid and for that we use a 25x objective um that is um mounted in our Blaze microscope and here we can get a better resolution right so you we can see clearly for example the nuclear staining and the projections of this um th positive cells that are going from the midbrain to to the Statum organoid and because of the better quality of the
• 49:30 - 50:00 image right we are able to do a segmentation and U we should be able to quantify and to validate this Negro statal pathway connectivity of course we did uh test with u more or uh or different um organoids and here in the video you can also see how the projections of this gfp positive and th positive cells are going from one um from one Organo to the other
• 50:00 - 50:30 inside of this assem um sample so we can identify these neurons that if you think uh that you have um a sparse label or or a few uh quantity of this or amount of these cells uh is is really important to to keep the and
• 50:30 - 51:00 to be able to scan the complete sample without cutting first because neurons could project in any direction so if you decide to cut your sample to analyze this you have a high chance to cut these projections and uh and lose the information of where is going um and if you have few cells then also if you are able to scan through the complete sample and then magnify and and and get um a
• 51:00 - 51:30 proper U resolution right then also you increase the chances of finding these neurons into the samp so as a summary what I would like to to to show you is that we have a a method that will work with really uh extended samples about 1 square cm more than 3 mm in thickness we are able to
• 51:30 - 52:00 have super resolution features what is important is that we we can preserve uh Auto proteins gfp M Cherry TD tomato M seruan and so one and it's compatible with commercial antibodies so if you have a sample right that you have to uh that that you already know how to prepare that you already have the antibodies then you don't have to change nothing just to apply this optimized protocol and bring it to the light sheet
• 52:00 - 52:30 in order to have the the a nice uh data and what is important is that with this right methodology we have we can image our sample over five orders of magnitude right without losing any information so that's probably the the the most important point that I would like that you keep in mind uh so you can go to different scales
• 52:30 - 53:00 during the same Imaging session and with this I would like to uh say thank you to to to my group work group the previous one where I was in the biophysical chemistry group and my new work group in the neuro connectomics with Professor Martin Schwartz in the uni clinicum here in in B so thank you and ready for questions thank you very much all for those
• 53:00 - 53:30 beautiful presentations we already have some questions coming in from the audience so we can get right to those so um first question is somebody who is a little bit less familiar with the topic and they're asking question do these organoids also mimic immune response to the drugs that are used for Parkinson's Disease is anybody from the panel able to address that question yeah I guess that goes to me um so mid brain organoids in their normal fashion in
• 53:30 - 54:00 their kind of first generation would not have the immune system because they derived from neuroectodermal stem cells so from the ectoderm immune system comes from the neoderm so we would have not we would have no immune system in there however I've showed you the second generation that also contains microa so it would derive them separately from IPS and then simply mix them in um and these indeed um mimic immune functions and um also these micro gler in there would for example respond to to drug treatment
• 54:00 - 54:30 yes thank you and next question is as the organoid models become increasingly sophisticated what are the ethical implications of using human derived tissues for research purposes and how can we ensure that the models are used responsibly and ethically um guess it goes again to me so on the one hand everything is derived human stem cell based right so there is a first first line of defense if you
• 54:30 - 55:00 will so we have to have obviously um aics approvals to work with these human stem cells and I can only talk about the national regulations we have here in Luxemburg so I have to present my project in front of an aics board and ask answer questions and get approval but then next step is obviously for organoids which are complex and then there's this whole idea that they der develop a level of self-awareness and these kind of things which I I would say we are pretty far away from that nevertheless this needs to be um eally
• 55:00 - 55:30 controlled I think it has to be overseen by an independent board um and I think the procedures at least what I can say for for our situation here are in place for that thank you and the next question is for Juan um regarding the sample preparation can you elaborate on the digestion process that makes the sample transparent yes sure um so the the the thees buffer is basically made uh to
• 55:30 - 56:00 work with prot SC so we have EDTA 1 T and Tron um and the idea is to use protein SK to break all the protein to protein interactions or protein to lipid interactions so everything that is not attached to the gel right by the Linker molecule will be washed will be washed away so that's why uh finally the the sample is transparent because we have basically all the lipids gone so that's
• 56:00 - 56:30 how we make the the the digestion and we try to always optimize um to the use of prote SK so prot SK is usually optimal at temperatures of 50 55 and a pH of8 so we don't uh we optimize the pH to that number but we don't do a digestion in a high temperature because that will degradate other proteins like for example Flores and proteins so we decrease the temperature to
• 56:30 - 57:00 37° so it's a mild digestion that maybe it will take a bit more uh long to to get the sample transparent but at the end you will be able to preserve more information and to destroy uh less of the proteins that also you would like to preserve thank you and the next question is can 3D imaging be used to observe Mouse brain regions as well yes um so I Ian about the the blade with
• 57:00 - 57:30 the blaz there are also other techniques not only expansion that you can use to clear the the tissue and depending on that uh right you can choose also between different objectives to match the different refractive index that these clearing methods uh could have like you have the organic solvents that usually have like a higher refractive index but uh in The Blaze you also are able to exchange um not necessarily the
• 57:30 - 58:00 complete objective but just the cap in order to match that those requirements uh so yeah I mean it's it's it's a really versatile tool if if you want to put a transparent sample under the light chip so it's even possible to image a whole Mouse so not only a brain or organoid but even a mouse thank you so Yen you touched on this a little bit maybe you can address this as well um
• 58:00 - 58:30 somebody's asking um you mentioned that the organoid recapitulates most of the midbrain cells the question is does the Organo also possess microglia yeah that pretty much is in line with my first answer right so um an organoid or midbrain organoid that derived from your epicate stem cell does not contain microa becomes it comes from the neao dermal lineage if but you can mix in micr gler later that you
• 58:30 - 59:00 separately derive from ipscs and that's by now actually the standard model we are using most of the time so yes you would have micr gler in there thank you and the next question is can the 3D imaging be used to oh no we answered this one already next one is in the expansion protocol did you test other types of endogenous Flores other than egfp whether these are preserved and whether it was preserved as well yes uh
• 59:00 - 59:30 so yes I I test different um endogenous Flor fores right like um efp TD tomato M Cherry M seruan um and I think that most of them are working fine usually when uh we have the problem that if there is just few a groups or no amine groups in that in that fluorescent
• 59:30 - 60:00 molecule that will not be preserved but it's possible to fix that if you change the Linker molecule right in order to catch one of the the one group that is present in the in this floresent molecule uh that's I would say the only restriction also then when you preserve this for example fluorescent molecule and after the digestion if you notice that the the intensity is not that High you still are able then to do a a post
• 60:00 - 60:30 digestion uh labeling antibody labeling because you will have right the signal of the of the fluoresent protein that it's a bit weak uh but that means that the epitopes is are available so you can use like a primary or secondary in order to boost that signal again thank you the next question is for yens there are some reports that have found some relationship between the pathological conditions and mechanical
• 60:30 - 61:00 properties of brain tissue question is have you explored anything in regards to this to better mimic the human pathological conditions in your organoids now that's a very interesting point we have not touched on that um so what we know is that in order to grow brain organoids we need relatively soft matrices like gel TRX or matrigel and if you get more stiff matrices that would not work but um we haven't done anything in the direction of pathology um although I fully agree it's interesting
• 61:00 - 61:30 but I cannot really answer anything on that thank you and the next question is how can uh patient derived ipscs be used to gener to generate personalized organoid models that accur accurately reflect the genetic and phenotypic heterogeneity of Parkinson's disease well a prior because it is PTI it is IPS derived it is patient specific so each model that we produce is by definition
• 61:30 - 62:00 personalized wonderful and another question which is a bit more technical can you please clarify whether using the four times objective for collection of fluorescence in this case is lateral resolution very low um well we use the 4X just to have like an overview um of the organoids and yes the resolution will be low with the 4X but you have to consider that the
• 62:00 - 62:30 resolution is always uh it's depending what do do you want to see right if we want to see a complete body right the complete organoid that is like almost like one more than one millimeter in size then the resolution of the 4X is good enough just to see the the complete U let's say elements right now when we show this high resolution images what we did to change to a higher magnification objective for the assembl loids was a
• 62:30 - 63:00 25x with a high numerical aperture so in order to have better resolution images so I maybe he was confused when because I was talking about four times or four four times expansion um but that's one thing so if you expand four times then you improve the resolution of your microscope by before uh and that's different from from the objective that you use so so just to make clear so we use yeah low resolution objectives to have like an overview or
• 63:00 - 63:30 to measure things in the mesos scale and then we can change to different magnifications to go to the nanoc scale thank you and uh another question we have is how long does it take um how long is the process from sample expansion uh for the Imaging so how long does it take to process but the the sample preparation for expansion U depends on how big is the
• 63:30 - 64:00 sample because you what is important is that the Linker molecule right penetrates the complete sample also this polyamid gel Matrix right that is the responsible for the mechanical expansion um so for example a sample imagine that is a sample of like a a sphere of one millimeter usually you have uh that sample the complete process will be like like 3 days more less to
• 64:00 - 64:30 have a completely transparent sample that will be ready to to be imaged uh under the light sheet now the Imaging on the light sheet is like I would say not more than one day uh because it's really fast and and I mean one day if you want to really go with several region of inter and and check with different magnifications thank you
• 64:30 - 65:00 um okay we have time for one more question question is how can we leverage Advanced 3D imaging techniques to bridge the gap between invitro organiz models and invivo human brain brain Imaging to provide a more comprehensive understanding of the spatio temporal dynamics of neurod degeneration and Parkinson's disease and I guess the followup to that is also to enable the development of new new Therapies ju that goes to
• 65:00 - 65:30 you that's uh well um well I think that all this so if we want to mimic right what is happening in in 3D I mean this 3D imag is completely necessary I mean that's it's already uh easy to see how how much like a model right is is changing right from a 2d culture to this organoids so I think that is uh oh that the question is gone um so um I think that
• 65:30 - 66:00 is really important especially to to be able to uh get the complete 3D information without altering the tissue I think that that is actually uh um um the most important part independent the method that you use to to make your sample transparent to image uh you need to always think in the Integrity of the sample right so I show you in one slide why we are using this technique is because we test enough and we are sure that we are preserving right the the 3D
• 66:00 - 66:30 morphology of the sample if you able to get that then you can start to translate that and maybe at some point right breach the knowledge uh between between this this tiny models to what is happening in a in a human brain also making the link with uh for example MRI imaging like functional Imaging and try to to to to merge all this information fantastic thank you so much
• 66:30 - 67:00 uh thank you to all of you for that discussion and for your wonderful presentations we appreciate you joining us here today thank you if anyone has any further questions please consider reaching out to the speakers directly their contact information is shown on the screen as a reminder the webinar will be archived on the scientist website you'll receive an email notifying you when the on demand webinar is available make sure to follow the scientists on social media for updates on future webinars I'd like to thank everyone who
• 67:00 - 67:30 took the time to join us today and especially those of you who shared your questions and comments on behalf of the scientists I'd also like to thank our speakers again Anisha Minos buul yans Christian schwamborn and Juan Eduardo Rodriguez gatia as well as our sponsor mueni biotech thank you everyone
• 67:30 - 68:00 [Music]