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

We are developing and optimizing organoids 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 special 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 are 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 the underlying mechanisms of human diseases such as Leigh syndrome and herpes simplex encephalitis. Furthermore, we developed a 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. To begin, collect up to five human brain organoids generated from induced pluripotent stem cells in one well of a 6 well plate. Aspirate excess culture media and wash the organoids once with PBS.

Using a scalpel, mince the organoids thoroughly. Add enzyme mix 1 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 1, 000 microliter pipette tip.

Then add enzyme mix 2 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 300g for 10 minutes at 4 degrees celsius. Aspirate the supernatant, and resuspend the pellet in 4 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 resuspend the pellet in NSB Plus medium according to a microfluidics-based scRNA-Seq kit manual. To begin, transfer four human brain organoids generated from induced pluripotent stem cells into a 6 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 2 milliliter douncer. Aspirate PBS completely and add 1 milliliter of NP40 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 1 milliliter of lysis buffer, and transfer the solution to a centrifuge tube. After 5 minutes of incubation at room temperature, centrifuge the organoid suspension at 500g for 5 minutes.

Aspirate the supernatant leaving 50 microliters in the tube. Add 1 milliliter of NSB Plus medium into the tube, and, after 5 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 500g for 5 minutes at 4 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.