Developing new tools to study and program cell fate at the single-cell level

Single-cell transcriptomics (scRNA-seq) started a technological revolution in biology by enabling through the plethora of methods to assess a molecular state of the cell on systems level without the strict necessity of the prior knowledge of the cell state. This wealth of data enabled us to answer fundamental problems of biology – how cells develop into plastic tissue, how they orchestrate developmental and homeostatic processes?

This thesis will cover developing novel methods and assays to study, map and perturb cell fates using droplet microfluidics-based scRNA-seq. First part will cover our re-engineering Drop-seq setup, for broader adoption and more efficient sample handling. The developments presented will cover a novel flexible workflow that significantly simplifies the sample handling while being efficient in single-cell material recovery.

Building on previous advances, the next part will cover a novel dropleting platform for deterministic mRNA capture bead and cell co-encapsulation (DisCo) and its application to study single intestinal organoid development. Here, a key advancement, so far unsolved in the field is ability to deterministically control the cell capture process. Although at lower throughput than other droplet microfluidics-based methods, this method allows processing low cell input samples with high capture efficiencies. As a proof-of-concept study, we have studied early development of single intestinal organoids. DisCo helped us in uncovering extensive organoid heterogeneity and to identify the three distinct and aberrant subtypes of intestinal organoids such as enterocysts, spheroids and gobloids.

Finally, the third part will cover the development of novel gain of function barcoded transcription factor (TF) screening in conjugation with scRNA-seq (TF-seq). To understand the role of TF in maintaining cell types, systematic manipulation of TFs combined by global gene expression profiling will aid in delineating their role in cell state progression and maintenance. We applied TF-seq on murine mesenchymal stem cell-like C3H10T1/2 model and found multiple known factors that program cells to adipose (Cebpa, Pparg), osteogenic (Runx2, Dlx3), and myogenic lineages (Myod, Myog). Additionally, we discovered and confirmed two novel TFs (Rhox12, Mycn) involved in adipogenesis. By analyzing both TF activity and genome-wide changes caused, this approach has the power to uncover both strong and weak drivers of lineage commitment among TFs.