Full-length, single-cell RNA-sequencing of human bone marrow subpopulations reveals hidden complexity

Bone marrow progenitor cell differentiation has frequently been used as a model for studying cellular plasticity and cell-fate decisions. Recent analysis at the level of single-cells has expanded knowledge of the transcriptional landscape of human hematopoietic cell lineages. Using single-molecule real-time (SMRT) full-length RNA sequencing, we have previously shown that human bone marrow lineage-negative (Lin-neg) cell populations contain a surprisingly diverse set of mRNA isoforms. Here, we report from single cell, full-length RNA sequencing that this diversity is also reflected at the single-cell level. From fresh human bone marrow unselected and lineage-negative progenitor cells were isolated by droplet-based single-cell selection (10xGenomics). The single cell-derived mRNAs were analyzed by full-length SMRT and short-read sequencing. In both samples we detected an average of 8000 different genes using short-read sequencing. Differential expression analysis arranged the single-cells of the total bone marrow into only four clusters whereas the Lin-neg population was much more diverse with nine clusters. mRNA isoform analysis of the single-cell populations using full-length sequencing revealed that Lin-neg cells contain on average 24% more novel splice variants than the total bone marrow cells. Interestingly, among the most frequent genes expressing novel isoforms were members of the spliceosome, e.g. HNRNPs, DEAD box helicases and SRSFs. Mapping the isoforms from all genes to the cell type clusters revealed that total bone marrow cells express novel isoforms only in a small subset of clusters. On the other hand, lineage-negative progenitor cells expressing novel isoforms were present in nearly all subpopulations. In conclusion, on a single-cell level lineage-negative cells express a higher diversity of genes and more alternatively spliced novel isoforms suggesting that cells in this subpopulation are poised for different fates. Graphical abstract