Focused ultrasound induced maturation of human brain organoids

Applicant: Minna-Mari Emilia Tervo, MSc, tervomin@uef.fi, University of Eastern Finland (UEF)

1. Overview

Organoids derived from human induced pluripotent stem cells (mini-brains) have become important models not only for studying neurodevelopmental and many other disorders, including Alzheimer's disease. One of the key unsolved problems in this continuously improving technology is the lack of key mechanosensitive signals that significantly contribute to early brain development in utero. The latter cues come from the pulsatile circulation of cerebrospinal fluid and blood and from increasingly growing tissue stiffness. The lack of these signals may contribute to the slow maturation of neuroglial cells and delayed formation of neural networks, as well as the chaotic/disorganized neuronal processes that make organoids significantly different from well-organized cortical layers. The key idea of this high-risk proposal is to use non-invasive focused ultrasound or specific chemical activation to promote better maturation and organization of mini-brains through the activation of calcium-permeable mechanosensitive channels such as Piezo1, which are known to be expressed in both glial and neuronal cells in the brain.

The proposed work is part of a PhD project and will be carried out in the Malm lab (UEF). All the needed equipment and cell lines are readily available in the lab. The work timeline is planned for a year, which will be performed in 2026-2027, starting in the fall of 2026. The proposed project involves the use of human induced pluripotent stem cells (iPSCs). The iPSC lines used in the study are obtained through collaboration. In order to protect the privacy and the confidentiality of the donor individuals, all identifiers associated with the donors have been removed. The donor consent has been obtained by my collaborators. The research data produced will be accessible to the research group after the PhD project is finished. The data will be published in international high-impact peer-reviewed journals with open access.

2. Introduction

This project represents a synthesis of three research lines, combining our expertise with human brain organoids, physiology and pharmacology of Piezo channels, as well as ultrasound technologies. Below these lines are presented in detail. This concept is novel and offers a mechanistic explanation (molecular targets) underlying the beneficial effects of this technology, which, if ultimately confirmed in the widespread expression of Piezo channels in developing cells, could become a common procedure in various types of organoids.

Organoids

Cortical iPSC derived organoids have emerged as important models for studies of neurodevelopmental and neurodegenerative disorders. Our laboratory has developed this technology and has made significant progress in refining models that facilitate the maturation and development of neural networks by fusing neuronal/astrocytic cell lines with microglia derived from different progenitors^1,2. However, a major drawback of organoid technology across all laboratories working in this field is the lack of naturally organized neural processes, making these networks fundamentally different from the well-organized neurons in the cortical layers of the human brain. Other limitations include lack of vascularization and pulsatile blood/ glymphatic flow. Thus, one of the reasons for slow maturation of organoids in this continuously improving technology is the lack of key mechanosensitive signals that significantly contribute to early brain development in utero.

Piezo

Mechanotransduction is a basic physiological property of all living species based on the high responsiveness to physical signals from the internal and external environment. This property is supported by wide expression in the plasma membrane of specific mechanosensitive ion channels. One of such recently discovered transducers is the mechanosensitive calcium-permeable Piezo1 ion channel is widely distributed in the central nervous system (CNS). Because of its high sensitivity, it plays a leading role in neuronal and glial mechanotransduction^3,4. Altered biomechanical properties, such as elevated stiffness in the brain tissue is common in many CNS disorders⁴,⁵ and could play a role of disease progression. The key role of Piezo1 channel for brain development is clear from the drastically smaller-sized brain in animals lacking this channel⁶.

FUS

While Piezo1 channels can be chemically activated with the selective agonists like Yoda1⁷ and Jedi molecules⁸, the mechanical stimulation of Piezo1 as a therapeutic approach is only emerging.

Transcranial focused ultrasound (FUS) has gained growing attention in both basic and clinical research due to its unique ability to non-invasively target deep brain regions with exceptional precision^9,10. I have been using FUS technology to activate cells in culture for the last 2 years and found that FUS can increase Ca2+ level in cells along with the generation of inward (depolarizing) membrane currents.

In support of our approach, recent study aiming to transplant organoid into the mouse brain¹¹ has showed a positive effect of FUS on organoid maturation but did not consider the role of membrane Piezo1 channels, which are central executors of FUS effect, for our project. These organoids also did not contain microglia, which derives from different progenitors and play a key role in brain maturation at early stage of neurodevelopment.

Here we hypothesize (Figure 1): (H1) that FUS stimulates proliferation of progenitors, promote mobility and maturation of cells and neuronal networks in the organoid and provides more aligned neuronal processes in a Piezo1-dependent manner; (H2) FUS will promote better coupling of glial cells with neurons to optimise neuronal maturation; (H3) specific Piezo1 agonist Yoda1 will provide beneficial effects similar to FUS and well be used both as a positive control and as a sensitizing agent for FUS.

Figure 1. FUS generates mechanical forces that stimulate mechanosensitive ion channel Piezo1.

To test these attractive hypotheses are specific aims are to: 1) to test the effect of low-intensity pulsed FUS stimulation on dendritic trees of neurons and neuronal network properties in iPSC -derived organoids in comparison with untreated and Yoda1-stimulated cells; 2) elaborate how FUS, acting via Piezo1, regulates glia-neuron interactions.

3. Methods

We will take advantage of the human-based models, induced pluripotent stem cell (iPSC)-derived microglia (iMGL)¹ and microglia containing organoids (iORGs)² developed in the Malm lab. Malm lab has demonstrated that the presence of microglia supports neuronal maturation in the iORGs². Thus, our iPSC technology provides an ideal human model for this study.

FUS application will be provided by our house-built system with a transducer located above the well plates with growing organoid samples. The pulse repetition frequency, which we have already found effective in microglia, is 1 kHz, with a 20% duty cycle and an acoustic pressure of 0.580 MPa. We plan to stimulate organoids for 5 minutes twice weekly for a total of 1 month, starting from day 60.

As readouts of efficient maturation of organoids treated via FUS/Yoda1 we will use: the length/alignment of the dendritic tree and number of dendritic spines, intensity of spontaneous and K+ induced spiking activity, especially in the gamma band, immunolabeling of neuronal, astrocytic, and microglial markers, and depolarizing action of GABA.

Participants/Sample

We will use organoid lines already available in the Malm lab, with and without microglia, to test the effect of ultrasound stimulation on the neuroglial properties of organoids. The iORG group size is 20/group, which according to the power analysis based on patch clamp data (power level 80%, mean difference 50%), is enough to show significant differences¹²

Materials and Procedures

Task 1. Effect of low-intensity pulsed FUS stimulation on dendritic trees of neurons and neuronal network properties in iORGs.

Aims. The aim here is to explore how repeatedly applied FUS at 1 kHz pulse repetition frequency (PRF) modulates neuronal growth and neuronal network properties in organoids.

Novelty. This will uncover the effect of FUS modulation of the maturation of neurons in human iORGs and the role of Piezo1 channels in this effect.

Methods. We will use our house-built FUS system, which is already installed in our lab and has shown efficiency in precluding Ca²⁺ signals and membrane depolarization in acute experiments (Tervo et al., manuscript in preparation notice Figure 3). Further details of the procedure are shown in Figure 2. The dendritic tree will be constructed either from biocytin-labelled (via patch pipette) samples or via 3D reconstruction in transparent organoids. Neuronal oscillations will be recorded by our micro-electrode array (MEA) -setup.

Expected results. We expect that FUS can increase the length of the dendritic tree and the number of dendritic spines, as well as provide better alignment of processes. Using MEA technology, routinely used in our lab, we will explore the appearance of spontaneous and K+ evoked network activity in particular in gamma band, important for memory consolidation. We will also test the connectivity properties of FUS-stimulated neuronal networks.

Risks and contingency plan. Main risks are related to finding optimal patterns of stimulation and the starting period for stimulation. To this end, our protocols for organoid stimulations are based on our acute FUS experiments, which have already shown the efficiency of FUS with a pulse repetition frequency of 1 kHz, 20 % duty cycle and acoustic pressure of 0.580 MPa. Involvement of Piezo1 in the modulation of neuronal networks will be tested with the specific Piezo1 agonist Yoda1. We will validate these using antagonist gadolinium chloride (Gd³⁺) and specific toxin GsMTx-4.

Figure 2. Method for FUS stimulations on a 24-well format. Organoids are placed temporarily in a 24-well plate (LUMOX, Sarstedt) for FUS stimulations. The wells are filled with medium, and the ultrasound transducer is placed in contact with the liquid. Ultrasound waves travel through the organoids and reach the absorber, which reduces reflections and standing waves, stabilizing the method.

Task. 2. Modulation by FUS glia-neuron interactions.

Aims. To establish the role of FUS-activated Piezo1 channels in the maturation of astrocytes and microglia in human organoids.

Novelty. We have already previously shown that activation of Piezo in 2D culture can activate microglial mobility⁵. Here, we explore this effect on astrocytes and microglia in 3D human mini-brains.

Methods. We will use FUS stimulations optimized in task 1. The organoids are analysed at 4 and 6 months old in culture and evaluated by immunohistochemistry (IHC) for neuronal maturation (TUJ1, MAP2, DCX, STMN2, GAP43, SOX11), diversification (reelin, Lhx6, GAD2, DLX1, DLX5), neuronal progenitors (TBR2), and proliferating (Ki67) and apoptotic (Annexin V) progenitors, microglia markers (Iba1), interneuron markers (parvalbumin, somatostatin) and astrocytes (GFAP, Aqp4, SOX9, FABP7, ID3, ID4, SLC1A3).

Expected results. Based on the labelling of neurons, astrocytes, and microglia, we will provide data on how FUS and Yoda1 modulate the development of astrocytes and their coupling with neurons, as well as the intensity of microglial maturation.

Risk and contingency plan. The risks are low as these labelling techniques are already established in our lab.

Planned Analyses

Analysis of dendritic trees and spines

The neurons are filled with biocytin via patch pipette to visualize their morphology. Reconstructions are performed with a calibrated XYZ axis Ludl electronics stage under a x100 oil objective of a Nikon Eclipse microscope using Neurolucida software (version 10, MBF Biosciences). Reconstructions are done for neurons showing largely preserved and intact apical and basal trees. Analysis of the morphological features (e.g. total dendritic length, total nodes, spine density, branching order) for the neuron is conducted via Neurolucida Explorer software available at the UEF as described previously¹³.

IHC analysis

Quantitative analysis of neuronal and microglial morphologies, microglia-neuron interaction, are done using Imaris setup.

MEA-analysis

To determine FUS effects on neuronal development at the network activity level, we analyse the human organoids with microelectrode array (MEA) recordings after FUS or control treatment. Data is acquired using the MEA2100-Mini-System (Multichannel Systems, Germany) which is optimal for organoids due to sample penetrating 3D electrodes². In our standard protocol activity is recorded in baseline conditions, under increased excitation (8 mM K⁺), and under increased excitation combined with blockade of inhibitory neurotransmission (100 µM picrotoxin), which allows assessing general excitation-inhibition balance of the network. Data is converted to standard hdf5-files and analysed with an in-house developed Python pipeline. Statistical analysis is performed with non-parametric tests (Kruskal-Wallis test, Mann-Whitney U-test). We predict that FUS promotes the development of neuronal network activity. Risk is low as the technique is in daily use in our lab. However, if we are unable to detect activity, we then analyse neuronal function at the single-cell level using patch clamp.

Statistical analysis

Statistical analyses are carried out using GraphPad Prism or SPSS. The ORG group size is 20/group, which according to the power analysis based on patch clamp data (power level 80%, mean difference 50%), is enough to show significant difference¹¹. All analyses are performed by investigators blinded to the study groups. The appropriate statistical analyses are performed using parametric and non-parametric tests depending on distribution with using GraphPad Prism (two-tailed t-test or Mann-Whitney and Kolmogorov-Smirnov respectively). Results are expressed as mean±SD and with P <0.05 are considered statistically significant.

Risk assessment

I do not expect critical obstacles in any part of the study. Contingency plan includes modification of FUS patterns and combination of FUS with chemical sensitization of cells by Piezo1 agonist Yoda1. Risks and if-then (plan B) also presented above in particular tasks. More detailed assessment of risks and a contingency plan is presented in the Table 1.

Risk (IF)	Risk	Mitigation/Alternative implementation (THEN)
Differentiation problems with iPSCs and ORG functionality	Low	Based on risk assessment analysis extra ORGs are grown taking into consideration that little fraction can be not functional / use of additional pairs of lines.

Table.1. Description of the possible risks in the study and alternative implementation.

Ethics and Data Management

The proposed project involves the use of human induced pluripotent stem cells (iPSCs). The iPSC lines used in the study are obtained through collaboration. The lines are already available and in culture in AIVI laboratory. In order to protect the privacy and the confidentiality of the donor individuals, all identifiers associated with the donors have been removed. The donor consent has been obtained by my collaborators. Written informed consent is available from all study participants. All materials have been collected and handled according to the national ethical guidelines and guidelines for Good Clinical Practice. Moreover, the novel human-based models used in this study decrease the need for animal studies supporting the 3R principles.

We follow the guidelines of the Finnish Advisory Board on Research Integrity, the recommendations on ethical review in human sciences, good scientific practice and valid legislation, including e.g. the Helsinki declaration in its latest version, GCP, and the General Data Protection Regulation (2016/679, GDPR) and guidelines set by the European Commission (European Communities Council Directive, 86/609/EEC). We comply to Convention of human rights and Biomedicine (Oviedo, 4 April 1997) regulations and EU Directive 2005/28/EC for good clinical practice.

The data will be stored and analyzed on password-protected secure servers, network drives, personal work computer, and portable hard drives. The data will be accessible to the research group after the PhD project is finished. The data will be published in international high-impact peer-reviewed journals with open access.

4. Pilot Data (Optional)

Figure 3. My preliminary results show FUS provides Ca2+ rise and depolarizing effect on microglia. FUS also promotes the uptake of FM1-43 dye, which was tested using the setup proposed in this plan. Calcium imaging (B) and electrophysiological methods (C) were used to investigate whether FUS responses in human microglia cell line are Piezo1-dependent. (A) Schematic of my established FUS setup for acute application. (B) Mean Ca2+ traces in microglia in control (no FUS) and FUS-treated cells (T= timepoint in control). YODA1 (Piezo1 agonist, 5 µM) was used to open Piezo1 channels. Stretch-activated calcium channels were blocked with 10 µM Gadolinium chloride (Gd3+), including Piezo1. (C) The FUS impact on microglia membrane potential was studied using voltage imaging. The mean voltage imaging traces show extracellular Ca2+ dependent membrane depolarization after FUS. (D) Schematic of my established FUS setup for 24-well plate. (E). Uptake of FM1-43 dye in microglia, control and FUS (2x60 s stimulation). YODA1 (5µM) was used as a positive control of FM1-43 uptake. Imaging 30 min post-FUS. Mann-Whitney, n=4-5. (Unpublished data.)

5. Budget

Organoid growth materials reagents (160 iORGs)	3200 €
LUMOX plates	500 €
Chemical compounds (Yoda, Gd3+, GmTx4)	500 €
Salary (80 %, PhD candidate)	17 800 €

Total: 22 000 €

6. References

1. Konttinen H, Cabral-da-Silva MEC, Ohtonen S, et al. PSEN1ΔE9, APPswe, and APOE4 Confer Disparate Phenotypes in Human iPSC-Derived Microglia. Stem Cell Reports. 2019;13(4):669-683. doi:10.1016/j.stemcr.2019.08.004

2. Fagerlund I, Dougalis A, Shakirzyanova A, et al. Microglia-like Cells Promote Neuronal Functions in Cerebral Organoids. Cells. 2021;11(1):124. Published 2021 Dec 30. doi:10.3390/cells11010124

3. Zheng Q, Liu H, Yu W, et al. Mechanical properties of the brain: Focus on the essential role of Piezo1-mediated mechanotransduction in the CNS. Brain Behav. 2023;13(9):e3136. doi:10.1002/brb3.3136

4. Li B, Zhao AR, Tian T, Yang X. Mechanobiological insight into brain diseases based on mechanosensitive channels: Common mechanisms and clinical potential. CNS Neurosci Ther. 2024;30(6):e14809. doi:10.1111/cns.14809

5. Zong B, Yu F, Zhang X, et al. Mechanosensitive Piezo1 channel in physiology and pathophysiology of the central nervous system. Ageing Res Rev. 2023;90:102026. doi:10.1016/j.arr.2023.102026

6. Nourse, J. L., Leung, V. M., Abuwarda, H., Evans, E. L., Izquierdo-Ortiz, E., Ly, A. T., Truong, N., Smith, S., Bhavsar, H., Bertaccini, G., Monuki, E. S., Panicker, M. M., & Pathak, M. M. (2022). Piezo1 regulates cholesterol biosynthesis to influence neural stem cell fate during brain development. The Journal of general physiology, 154(10), e202213084. https://doi.org/10.1085/jgp.202213084

7. Jäntti H, Sitnikova V, Ishchenko Y, et al. Microglial amyloid beta clearance is driven by PIEZO1 channels. J Neuroinflammation. 2022;19(1):147. Published 2022 Jun 15. doi:10.1186/s12974-022-02486-y

8. Bryniarska-Kubiak, N., Kubiak, A., & Basta-Kaim, A. (2023). Mechanotransductive Receptor Piezo1 as a Promising Target in the Treatment of Neurological Diseases. Current neuropharmacology, 21(10), 2030–2035. https://doi.org/10.2174/1570159X20666220927103454

9. Miller DL, Smith NB, Bailey MR, et al. Overview of therapeutic ultrasound applications and safety considerations. J Ultrasound Med. 2012;31(4):623-634. doi:10.7863/jum.2012.31.4.623

10. Meng Y, Hynynen K, Lipsman N. Applications of focused ultrasound in the brain: from thermoablation to drug delivery. Nat Rev Neurol. 2021;17(1):7-22. doi:10.1038/s41582-020-00418-z

11. Li, X. H., Guo, D., Chen, L. Q., Chang, Z. H., Shi, J. X., Hu, N., Chen, C., Zhang, X. W., Bao, S. Q., Chen, M. M., & Ming, D. (2024). Low-intensity ultrasound ameliorates brain organoid integration and rescues microcephaly deficits. Brain : a journal of neurology, 147(11), 3817–3833. https://doi.org/10.1093/brain/awae150

12. Brunner, J. W., Lammertse, H. C. A., van Berkel, A. A., Koopmans, F., Li, K. W., Smit, A. B., Toonen, R. F., Verhage, M., & van der Sluis, S. (2023). Power and optimal study design in iPSC-based brain disease modelling. Molecular psychiatry, 28(4), 1545–1556. https://doi.org/10.1038/s41380-022-01866-3

13. Mohan, H., Verhoog, M. B., Doreswamy, K. K., Eyal, G., Aardse, R., Lodder, B. N., Goriounova, N. A., Asamoah, B., B Brakspear, A. B., Groot, C., van der Sluis, S., Testa-Silva, G., Obermayer, J., Boudewijns, Z. S., Narayanan, R. T., Baayen, J. C., Segev, I., Mansvelder, H. D., & de Kock, C. P. (2015). Dendritic and Axonal Architecture of Individual Pyramidal Neurons across Layers of Adult Human Neocortex. Cerebral cortex (New York, N.Y. : 1991), 25(12), 4839–4853. https://doi.org/10.1093/cercor/bhv188

Focused ultrasound induced maturation of human brain organoids

Focused ultrasound induced maturation of human brain organoids

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