Stem cell research: New approach in quantitative imaging cytometry

Daniel Coutu, Konstantinos Kokkaliaris and Leo Kunz from the research group of Professor Timm Schroeder recently developed an integrated pipeline that facilitates whole-organ quantitative imaging cytometry in three dimensions. They developed this approach to analyse bone and bone marrow, which are notoriously difficult to process for imaging. But why notoriously difficult?

Imaging of large organs as a whole and at single cell resolution in a quantitative manner remains challenging – why?

To put it simply, it’s the dimensions. It requires time and attention to details to prepare large samples while imaging them is time-consuming. Another and often overlooked challenge is that large organs just don’t handle as well as smaller tissues. Correct tissue preparation and handling in a way that the inherent tissue architecture is maintained without introducing imaging artefacts is crucial for doing deep-tissue quantitative imaging with single-cell resolution. Lastly, dealing with large imaging datasets remains computationally intensive.

You recently developed an integrated pipeline that facilitates whole-organ quantitative imaging cytometry in three dimensions. Could you explain how this integrated pipeline looks like and operates?

Our pipeline covers all steps from sample harvesting to image analysis. It starts with tissue dissection and fixation followed by tissue sectioning and multicolor immunostaining. An optical clearing method is described and settings for 3D confocal imaging of up to eight-colors are shown. After using commercial software to segment cells and structures of interest we show how to perform further analyses in our own software, XiT. We provide detailed protocols, optimization workflows and troubleshooting guides for each step of the pipeline.

With this new approach you analyse bone and bone marrow – why do you focus on bone and bone marrow?

Bone and marrow are home to adult blood and skeletal stem cells (the two stem cells most widely used in clinical trials). Despite extensive studies, the cellular and molecular composition of these tissues is controversial, mainly because of their structural complexity and hurdles associated with sectioning hard bone tissue enclosing the mostly liquid marrow, without distorting their architecture or introducing imaging artefacts. Our pipeline, designed to reproducibly analyze the most difficult organ, is easily applied to other tissues.

Your recent publications are valuable resources that will help clarify outstanding questions in bone biology, hematopoiesis, vascular biology, neurosciences and extracellular matrix research. Could you please specify – how will your findings help clarify outstanding questions in these medical fields?

We aimed at democratizing multicolor, 3D imaging cytometry so any scientist can reliably and reproducibly answer important questions in their respective fields. We also offer the community open-access imaging data of over 45 cellular and molecular markers expressed in bone or marrow tissues (an imaging atlas of bone, cartilage, blood vessels and pericytes, stroma, neurons and extracellular matrix). We list over 250 antibodies that have been tested in our pipeline so that scientists already know which ones to use for their specific questions.