isolation of single nuclei from human brain organoids for single-nucleus rna sequencing

Extração de células únicas e núcleos de organoides cerebrais humanos

Over the last years, organoid technologies have emerged as a promising tool to culture organ-like tissues1,2,3. Especially for organs that cannot be easily accessed, such as the human brain, organoids offer the opportunity to gain insights into&#160;development and disease manifestation4. As such, brain organoids have been widely used as an experimental model to investigate various human brain disorders, including developmental, psychiatric, or even neurodegenerative diseases4,5,6.
With the advent of single-cell transcriptome profiling technologies, primary human tissues and complex in vitro models could be studied with an unprecedented level of granularity, providing mechanistic insights into gene expression changes on the level of cell subpopulations in health and disease and informing about new putative therapeutic targets7,8,9. The organoid field has progressed by utilizing single-cell transcriptome profiling to assess cellular composition, reproducibility and the fidelity of brain organoid technologies10,11,12. Single-cell RNA-sequencing (scRNA-seq) enabled cell classification and the identification of genetic dysregulation in diseased organoids13,14. Importantly, it is the complexity of organoid tissues that necessitates the implementation of techniques that enable the profiling of individual cells. Characterization of organoids using methods such as bulk transcriptome profiling (bulk RNA sequencing) leads to masked cellular heterogeneity and gene expression profiles which are averaged across all types of cells within the complex tissue, ultimately limiting our understanding of ongoing processes during organoid development in health and disease15,16,17. As scRNA-seq methods continue to advance, an increasing number of atlases are being created, exemplified by resources like the Allen Brain Atlas or the Single cell atlas of human brain organoids by Uzquiano et al.18.
Accomplishing successful scRNA-seq from brain organoids relies on effective isolation and capture of intact cells. As the dissociation of brain organoids to obtain individual cells is based on enzymatic digestion, it can influence gene expression patterns by inducing stress and cell damage19,20. Hence, the dissociation of the tissue into individual cells is the most crucial step. An alternative approach is single-nucleus RNA sequencing (snRNA-seq), which facilitates the enzyme-free extraction of nuclei from both, fresh and frozen, tissue21,22. However, the isolation of nuclei from a tissue poses other challenges such as the enrichment of cell types of interest and the low RNA content of nuclei in comparison to cells.
Transcriptome studies of brain organoids are commonly conducted using scRNA-seq10,18,23. However, the isolation of single nuclei might provide an orthogonal and supplemental method to investigate the transcriptomic profile of organoids. Here, we introduce a toolbox for scRNA- and snRNA-seq for brain organoids and discuss the critical points for obtaining the best quality sequencing data.

Autor em destaque: Integrando modelos organoides com tecnologias de transcriptômica espacial e de célula única

Geração e análise a jusante de transcriptomas de célula única e núcleo único em organoides cerebrais

We are developing and optimizing organoid models in order to apply them for disease modeling. Our goal is to understand the molecular mechanism underlying the disease development and progression, and we are working in close collaboration with Nikolaus Rajewsky Lab, who is developing and applying the single-cell and spatial technologies in order to understand how the RNA regulate the gene expression in health and disease. In recent years, brain organoids have emerged as experimental and translational platforms for studying patient-specific diseases.Therefore, it's not unsurprising that most recent developments aimed at improving the complexity of brain organoid models. For instance, we are now able to generate brain region-specific organoids and also use brain organoids for multi-organoid assemblies. Also, various methods have been implemented to introduce vascularization and immune cells into brain organoids.Currently, one of the most research-advancing technologies that we're using in this lab is spatial transcriptomics, which allows the assessment of gene expression within intact tissue. This is crucial in understanding how gene expression profiles of different cell types vary in different regions within one tissue. Another recent advancement that has been made is single-cell multiomics, which allows the analysis of multiple modules of a single cell, which gives a more holistic approach in analyzing these cells.We employ brain organoids and single-cell and spatial transcriptomics methods to understand underlying mechanisms of human diseases such as Leigh syndrome and herpes simplex encephalitis. Furthermore, we developed high-resolution and cost-effective spatial transcriptomics methods called Open-ST. We compared and optimized the single-cell and single-nucleus RNA sequencing method to understand how well they perform regarding cell type recovery in brain organoid tissue.

Isolamento de Núcleos Únicos de Organoides do Cérebro Humano para Sequenciamento de RNA de Núcleo Único

Para começar, transfira quatro organoides cerebrais humanos gerados a partir de células-tronco pluripotentes induzidas para uma placa de seis poços contendo PBS resfriado. Corte cada organoide em quatro pedaços. Usando uma tesoura, corte a ponta de uma ponta de pipeta de 1.000 microlitros e use-a para transferir pedaços organoides para um douncer de dois mililitros.Aspire PBS completamente e adicione um mililitro de tampão de lise NP-40. Aplique os organoides três vezes com o pilão A seguido do pilão B. Transfira a suspensão para um tubo de centrífuga de 15 mililitros. Lave o douncer com um mililitro de tampão de lise e transfira a solução para um tubo de centrífuga.Após cinco minutos de incubação à temperatura ambiente, centrifugar a suspensão organoide a 500 G durante cinco minutos. Aspirar o sobrenadante deixando 50 microlitros no tubo. Adicione um mililitro de meio NSB Plus no tubo.E depois de cinco minutos, misture para ressuspender o pellet. Depois de preparar um gradiente de Percoll no tubo, coloque a suspensão organoide em cima dele. Em seguida, centrifugue a suspensão organoide em camadas a 500 G por cinco minutos a quatro graus Celsius.Aspirar o sobrenadante e ressuspender o sedimento em meio NSB Plus. Filtre a solução de núcleos em um filtro de células de 40 mícrons. Em seguida, corar os núcleos usando DAPI e contá-los em uma câmara de Neubauer.Centrifugue a quantidade necessária de solução de núcleos e ressuspenda o pellet em meio NSB Plus de acordo com um manual do kit snRNA-seq baseado em microfluídica.

isolation-single-nuclei-from-human-brain-organoids-for-single-nucleus

Research

JoVE Journal

Developmental Biology

Isolation of Single Nuclei from Human Brain Organoids for Single-Nucleus RNA Sequencing

133 visualizações. Para começar, transfira quatro organoides cerebrais humanos gerados a partir de células-tronco pluripotentes induzidas para uma placa de seis poços contendo PBS resfriado. Corte cada organoide em quatro pedaços. Usando uma tesoura, corte a ponta de uma ponta de pipeta de 1.000 microlitros e use-a para transferir pedaços organoides para um douncer de dois mililitros.Aspire PBS completamente e adicione um mililitro de tampão de lise NP-40. Aplique os organoides três vezes com o pi...

Vídeo: Isolamento de Núcleos Únicos de Organoides do Cérebro Humano para Sequenciamento de RNA de Núcleo Único

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparation&#160;and sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.

Here, we introduce a comprehensive protocol for the generation and downstream analysis of human brain organoids using single-cell and single-nucleus RNA sequencing.

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene ...

Biologia do Desenvolvimento

Isolation of Single Cells from Human Brain Organoids for Single-Cell RNA Sequencing

To begin, collect up to five human brain organoids generated from induced pluripotent stem cells in one well of a six well plate. Aspirate excess culture media, and wash the organoids once with PBS. Using a scalpel, mince the organoids thoroughly.Add enzyme mix one to the minced organoids, and incubate at 37 degrees Celsius with 20%oxygen and 5%carbon dioxide at 90 RPM for 10 to 15 minutes. Mix the organoid suspension using a 1000 microliter pipette tip. Then add enzyme mix two, and incubate at 37 degrees Celsius for 10 to 15 minutes.Stop dissociation by adding 10 milliliters of stop solution. Filter the cell suspension on a 70-micron cell strainer. Centrifuge the filtered cell suspension at 300 G for 10 minutes at four degrees Celsius.Aspirate the supernatant, and re-suspend the pellet in four milliliters of cold 0.04%BSA. Now filter the cell suspension on a 40-micron cell strainer, and count the cells on a cell counter. Centrifuge the required amount of cell suspension, aspirate the supernatant, and re-suspend the pellet in NSB plus medium according to a microfluidics-based scRNA-Seq kit manual.

Isolamento de células únicas de organoides cerebrais humanos para sequenciamento de RNA de célula única

To begin, transfer four human brain organoids generated from induced pluripotent stem cells into a six-well plate containing chilled PBS. Cut each organoid into four pieces. Using scissors, cut the tip of a 1, 000 microliter pipette tip and use it to transfer organoid pieces into a two milliliter douncer.Aspirate PBS completely and add one milliliter of NP-40 lysis buffer. Dounce the organoids three times with dounce pestle A followed by pestle B.Transfer the suspension into a 15 milliliter centrifuge tube. Wash the douncer with one milliliter of lysis buffer and transfer the solution to a centrifuge tube.After five minutes of incubation at room temperature, centrifuge the organoid suspension at 500 G for five minutes. Aspirate the supernatant leaving 50 microliters in the tube. Add one milliliter of NSB Plus medium into the tube.And after five minutes, mix to resuspend the pellet. After preparing a Percoll gradient in the tube, layer the organoid suspension on top of it. Next, centrifuge the layered organoid suspension at 500 G for five minutes at four degrees Celsius.Aspirate the supernatant and resuspend the pellet in NSB Plus medium. Filter the nuclei solution on a 40 micron cell strainer. Then, stain the nuclei using DAPI and count them in a Neubauer chamber.Centrifuge the required amount of nuclei solution and resuspend the pellet in NSB Plus medium according to a microfluidics based snRNA-seq kit manual.