Solana Group Stem Cell Biology and Evolution All multicellular organisms have pluripotent cells that can differentiate into multiple cell types. In animals, these are present in embryos but sometimes as well in the adult stages. Often this happens in animals that can regenerate and even use this capacity for asexual reproduction. Our group is interested in stem cells, pluripotency, differentiation, and their evolution across the animal tree of life. There are several conserved features in invertebrate stem cells, including RNA binding proteins and epigenetic regulators. However, in most animal groups the identity and properties of their stem cells remain unclear, as their study has been challenging for decades. Recently, single cell analysis has changed this. We have used single cell transcriptomics (scRNA-seq) to profile stem cell differentiation trajectories in the planarian Schmidtea mediterranea. Now, we aim at using single cell analysis across the animal tree of life to decode the basic principles of animal stem cells and regeneration. Our methodology We have developed a novel cell dissociation approach (ACME, García-Castro et al. Genome Biology) which fixes the cells while they are being dissociated, preventing the stress imposed by enzymatic live dissociation. ACME is very versatile and can be used in different species. We have also optimised a modified version of SPLIT-seq, a scRNA-seq technique that uses combinatorial barcoding. SPLIT-seq is cheaper than droplet methods, can be scaled up and allows multiplexing (avoiding batch effects). This enables us to perform functional genomics comparisons with cell type resolution. Our group uses the planarian model as a workhorse to develop new single cell methods to decode stem cell and differentiation properties, which can be later used in other animal stem cell models. ACME Dissociation (left) + SPLiT-seq (right): We have developed a novel approach for single cell transcriptomics that fixes the cells from the very beginning (ACME) and combined this with SPLiT-seq, a novel combinatorial barcoding method of single-cell transcriptomics that allows sequencing >50K cells in one run (Garcia-Castro et al. Genome Biology 2021). Studying stem cells functionally to learn their regulatory code The high multiplexing capacity of SPLiT-seq solves the problem of multisample experiments with single cell resolution. Our current technology can multiplex 2-24 samples in a single SPLiT-seq experiment, and we are upscaling this. This reduces batch effects, eliminating the need for integration approaches and dramatically reduces the price. For instance, we have used this multiplexing capacity to study cell type allometry using single cell transcriptomics – (Emili et al, bioRxiv 2023). By comparing planarians of different sizes we have measured changes in cell type composition, gene expression and cell size. We are currently upscaling this to study the role of transcription and epigenetic factors in stem cells and differentiation, as well as the molecular and cellular detail of planarian regeneration. Studying gene regulation with single cell resolution: Multiplexed approaches to single cell transcriptomics such as SPLiT-seq allow performing differential gene expression analyses with cell type resolution to investigate the role of individual genes in individual cell types. Stem cell biology and regeneration across the animal tree of life We aim at using methods developed in S. mediterranea to other regenerating organisms across the animal tree of life, to understand the cellular and molecular principles of stem cells and regeneration. Our laboratory has already generated a cell type atlas of the regenerating annelid Pristina leidyi and is generating cell type atlases of the cnidarian Hydractinia symbiolongicarpus (in collaboration with Uri Frank, Galway) and the colonial ascidian Botryllus schlosseri (with Lucia Manni, Padova). We are under way of obtaining other regenerating species. The long-term goal of our research group is to apply single cell methods to diverse regenerating species.