Single-cell RNA sequencing for subtype discovery in Plasmodium falciparum and mammalian cells

Since the dawn of massively parallel sequencing technologies in mid-2000s their utility in profiling the expression of genes in a genome-wide fashion has matured and progressed from cell populations to individual cells. In particular, single-cell RNA sequencing (scRNA-seq) has impacted numerous domains in life sciences and hold immense promise in biology and medicine. Indeed, it has become realistic to chart the complete set of cell types and states in multicellular organisms, and projects have started to map out cell types in humans (i.e. the Human Cell Atlas project) and model organsims. In this thesis, I present the application of scRNA-seq to infectious disease and cancer as well as a computational assessment of the general possibilities and limitations of scRNA-seq for enumerating cell types and states de novo.

In Paper I, we describe the ability of scRNA-seq to profile transcriptomes from individual malaria-causing P. falciparum parasites. We reveal heterogeneity even among synchronized cultures of parasites during their red blood cell life cycle. Moreover, we identify a subset of sexually differentiated P. falciparum with a distinct gene signature, likely important for parasite transmission that may be exploited for the design of transmission-blocking drugs and/or vaccines. In Paper II, I present a computational strategy to identify the magnitude of biological gene expression differences needed for accurate inference of cell identities using scRNA-seq. Interestingly, rather large differences are needed for proper cell state discrimination, irrespective of scRNA-seq protocol, implying that large number of cell states may escape detection. In Paper III, we used scRNA-seq and bulk RNA-seq to characterize the molecular programs during the later stages of lung metastasis. We demonstrate that a transition from epithelial to mesenchymal cell characteristics occurs in cancer cells during metastasis, and that the mesenchymal properties are maintained during metastasis growth extending over a week. In Paper IV we performed transcriptome analyses on stem and progenitor populations in myelodysplastic syndrome (MDS) patients. We provide evidence that the MDS stem cells and the progenitors have distinct transcriptome.

Altogether, this thesis expands the applications of scRNA-seq towards parasite biology and cancer metastasis and we provide valuable insights into the abilities of current scRNA-seq technologies in mapping cell states in an unbiased fashion.

List of scientific papers

I. Mtakai Ngara, Mia Palmkvist, Sven Sagasser, Daisy Hjelmqvist, Åsa K. Björklund, Mats Wahlgren, Johan Ankarklev and Rickard Sandberg. Exploring parasite heterogeneity using single-cell RNA-seq reveals a gene signature among sexual stage Plasmodium falciparum parasites. Experimental Cell Research. 2018 Oct 1; 371(1): 130-138.
https://doi.org/10.1016/j.yexcr.2018.08.003

II. Mtakai Ngara and Rickard Sandberg. Defining limits to subtype discovery in single-cell RNA-sequencing experiments. [Manuscript]

III. Azadeh Nilchian, Mtakai Ngara, Åsa Segerstolpe, Vedrana Tabor, Mikael Karlsson, Rickard Sandberg and Jonas Fuxe. Single-cell sequencing reveals autocrine TGF-b-induced EMT as a promoter of late-stage metastasis. [Manuscript]

IV. Petter S. Woll, Una Kjällquist, Onima Chowdhury, Helen Doolittle, David C. Wedge, Supat Thongjuea, Rikard Erlandsson, Mtakai Ngara, Kristina Anderson, Qiaolin Deng, Adam J. Mead, Laura Stenson, Alice Giustacchini, Sara Duarte, Eleni Giannoulatou, Stephen Taylor, Mohsen Karimi, Christian Scharenberg, Teresa Mortera-Blanco, Iain C. Macaulay, Sally-Ann Clark, Ingunn Dybedal, Dag Josefsen, Pierre Fenaux, Peter Hokland, Mette S. Holm, Mario Cazzola, Luca Malcovati, Sudhir Tauro, David Bowen, Jacqueline Boultwood, Andrea Pellagatti, John E. Pimanda, Ashwin Unnikrishnan, Paresh Vyas, Gudrun Go ̈hring, Brigitte Schlegelberger, Magnus Tobiasson, Gunnar Kvalheim, Stefan N. Constantinescu, Claus Nerlov, Lars Nilsson, Peter J. Campbell, Rickard Sandberg, Elli Papaemmanuil, Eva Hellström-Lindberg, Sten Linnarsson and Sten Eirik W. Jacobsen. Myelodysplastic Syndromes Are Propagated by Rare and Distinct Human Cancer Stem Cells In Vivo. Cancer Cell. 2014 June 16; 25(6):794-808.
https://doi.org/10.1016/j.ccr.2014.03.036