Scalable Approaches for Inferring Chromatin States and Lineages of Human Cells

Abstract

The human hematopoietic system is a paradigm for stem cell biology wherein a heterogeneous tissue (blood) is established and maintained by a small pool of stem and progenitor cells. Herein, this dissertation represents a collection of new approaches, both computational and technical, to chart cell fate transitions and clonal properties of the hematopoietic system. I present specific innovations that enable the massive-scale inference of chromatin accessibility in single cells as well as their clonal relatedness within humans. Importantly, these concepts, technologies, and innovations are broadly applicable to understanding human tissue biology in other systems. Chapter 1 introduces the concept of charting lineal relationships between cells (i.e lineage tracing) in human tissue by utilizing somatic mitochondrial DNA (mtDNA) mutations as clonal markers via single-cell genomics technologies. Further, I show that this concept enables scalable lineage tracing at a greater throughput (~1,000x) than other approaches for human cells. In Chapter 2, I demonstrate that somatic mtDNA mutations can be propagated longitudinally in vivo over ~3 years and in lineage-restricted progenitors. Together, these chapters provide the theoretical basis for scalable lineage tracing of hematopoietic cells. Next, Chapter 3 introduces a droplet microfluidics platform that enables profiling accessible chromatin in hundreds of thousands of single cells. I show how this approach can be utilized to dissect multi-lineage non-coding regulatory logic of hematopoietic tissue in response to stimuli. In Chapter 4, I identify and correct a previously uncharacterized artifact termed ‘barcode multiplets’ in single-cell data. Importantly, I show that, if uncorrected, barcode multiplets artificially inflate clonality estimates. These two chapters provide a technical basis for accurate, large-scale profiling and clonal estimation of human cells. Chapter 5 synthesizes these advances (mtDNA-based lineage tracing and droplet-based single-cell genomics) into one assay, termed mtscATAC-seq. Importantly, this multimodal approach provides a technical basis to simultaneously infer both contemporary cell state (via accessible chromatin) and cell fate (via somatic mutation lineage tracing), altogether enabling the dissection of complex tissues and stem cell hierarchies in vivo. Taken together, this body of work summarizes several key advances that uniquely enable the study of developmental and regenerative processes in native human tissue.