Spatially resolved transcriptomics (SRT) approaches have allowed for the investigation of transcriptomic defined cellular diversity of biological tissues previously unseen. A multitude of different SRT technologies have been developed over the years, addressing the various needs of the scientific community by enabling the characterization of molecular signatures in situ, while preserving tissue morphology.

Despite the multitude of SRT techniques developed, there is still no single ‘best’ SRT approach, due to trade offs different techniques have to compromise on. The SRT quadrilemma, termed in my thesis – Throughput, Specificity, Sensitivity and Multiplexing are the main characteristics that the dream SRT should possess, but is theoretically impossible due to the mutually exclusive nature of these characteristics. The work in this thesis focuses on the development of In Situ Sequencing (ISS) with padlock probes and rolling circle amplification, tackling the SRT quadrilemma.

In paper I, we investigate the efficiency bottlenecks of cDNA-HybISS against a commercial kit that targets RNA directly, circumventing cDNA synthesis in situ. We found that by circumventing cDNA synthesis in situ, we are able to improve the detection efficiency 5 fold. In addition, the increase in sensitivity enhanced analytical capability of our data and allowed for low, 5X magnification imaging.

In Paper II, we provide an end to end in situ transcriptomic solution with a RNA targeting ISS chemistry with improved detection efficiency compared to cDNA-HybISS and user friendly and well documented computational tools for probe design, image registration, decoding and analysis. In addition, we also demonstrate that our RNA-ISS is compatible with posterior stainings such as multiplexed antibody staining, opening up the possibility of spatial multi-omics all while maintaining cost effectiveness, customizability and ease of implementation of RNA-ISS.

In paper III, we show that we are able to achieve single nucleotide specificity with RNA targeted ISS. We show that we are able to distinguish human and mouse cells from the genotyping experiment with competing padlock probes targeting a conserved region of human and mouse beta-actin sequence that differs by a single base. In addition to the improved detection efficiency we show that the specificity with single nucleotide RNA-ISS is comparable to the established cDNA-BaSSIS method.

In paper IV, we further developed a RNA gap filling approach for genotyping. Here, we leveraged on a polymerase mediated approach for sequence capture, reverse transcribing a stretch of unknown sequences on RNA into the probe before ligation, amplification and sequencing readout. We demonstrate that we are able to fill a gap of 20nt with high fidelity as a first proof of concept experiment.

Lastly, in paper V, we employed targeted cDNA-HybISS for a proof of concept study as a high throughput molecular screening tool for a cohort study of control and schizophrenic post mortem study of the prefrontal cortex. We attempt to map cell type compositions and macroscopic tissue organization within this cohort as an exploratory study.

The work in this thesis presents the development of next generation in situ sequencing with improved sensitivity, specificity, throughput and multiplexing as a next generation molecular tool for spatial mapping of molecular signatures within biological samples in health and disease.

Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in situ expression maps of hundreds of genes. We use direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high-throughput and large multiplexing. We validate the method across a number of species and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental and tissue biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to high-throughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.

Understanding the rapidly evolving landscape of single-cell and spatial omic technologies is crucial for advancing biomedical research and drug development. We provide a living review of both mature and emerging commercial platforms, highlighting key methodologies and trends shaping the field. This review spans from foundational single-cell technologies such as microfluidics and plate-based methods to newer approaches like combinatorial indexing; on the spatial side, we consider next-generation sequencing and imaging-based spatial transcriptomics. Finally, we highlight emerging methodologies that may fundamentally expand the scope for data generation within pharmaceutical research, creating opportunities to discover and validate novel drug mechanisms. Overall, this review serves as a critical resource for navigating the commercialization and application of single-cell and spatial omic technologies in pharmaceutical and academic research.

We present a spatial omics approach that combines histology, mass spectrometry imaging and spatial transcriptomics to facilitate precise measurements of mRNA transcripts and low-molecular-weight metabolites across tissue regions. The workflow is compatible with commercially available Visium glass slides. We demonstrate the potential of our method using mouse and human brain samples in the context of dopamine and Parkinson’s disease.

The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5–12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.

Oligodendrogenesis in the human central nervous system has been observed mainly at the second trimester of gestation, a much later developmental stage compared to oligodendrogenesis in mice. Here, we characterize the transcriptomic neural diversity in the human forebrain at post-conception weeks (PCW) 8–10. Using single-cell RNA sequencing, we find evidence of the emergence of a first wave of oligodendrocyte lineage cells as early as PCW 8, which we also confirm at the epigenomic level through the use of single-cell ATAC-seq. Using regulatory network inference, we predict key transcriptional events leading to the specification of oligodendrocyte precursor cells (OPCs). Moreover, by profiling the spatial expression of 50 key genes through the use of in situ sequencing (ISS), we identify regions in the human ventral fetal forebrain where oligodendrogenesis first occurs. Our results indicate evolutionary conservation of the first wave of oligodendrogenesis between mice and humans and describe regulatory mechanisms involved in human OPC specification.