Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation

Abstract

In the current understanding of adult bone marrow hematopoiesis, megakaryocytes (MKs) originate from cells immuno-phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. Here, we use single cell RNA sequencing to characterize HSCs and MKs from human bone marrow, to investigate MK lineage commitment and maturation. We identify two MK primed HSC clusters exhibiting unique differentiation kinetics, at least one of which is used in steady state and stress thrombopoiesis. By analyzing transcriptional signatures we show that human bone marrow MKs originate from MK primed HSC subpopulations, supporting the notion that these display exclusive priming for MK differentiation. We show that transcriptional programs change with increasing MK ploidy, where genes upregulated in high ploidy states may have functional relevance in platelet production. Finally, we highlight the presence of a specific transcriptional signature in MKs from individuals with myocardial infarction, supporting the aberration of MK differentiation in this thrombotic state.

Benchmarking full-length transcript single cell mRNA sequencing protocols

“…The key divergence of the original SMART-seq2 method, is a step separating mRNA from genomic DNA (gDNA), into distinct single cell samples eligible for a range of library preparation protocols. This step also serves as an RNA purifying step, removing cell debris, protein and gDNA from the downstream reaction, and the method has been applied to hard-to-sequence cells, where the DNA fraction has been discarded [19]. The protocol originally applied a modified SMART-seq2 protocol for transcriptome amplification, and PicoPLEX or Multiple displacement amplification (MDA) for genome amplification [13,17].…”

Benchmarking full-length transcript single cell mRNA sequencing protocols

“…The key divergence of the original SMART-seq2 method, is a step separating mRNA from genomic DNA (gDNA), into distinct single cell samples eligible for a range of library preparation protocols. This step also serves as an RNA purifying step, removing cell debris, protein and gDNA form the downstream reaction, and the method have been applied to hard-to-sequence cells, where the DNA fraction has been discarded (Choudry et al, 2020). The protocol originally applies a modified SMART-seq2 protocol for transcriptome amplification, and PicoPLEX or Multiple displacement amplification (MDA) for genome amplification (Iain C. Macaulay et al, 2016;Simone Picelli et al, 2014).…”