Abstract
BackgroundHematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages. HSCs are typically purified from the bone marrow using cell surface markers. Recent studies have identified significant cellular heterogeneities in the HSC compartment with subsets of HSCs displaying lineage bias. We previously discovered that the transcription factor Bcl11a has critical functions in the lymphoid development of the HSC compartment.ResultsIn this report, we employ single-cell transcriptomic analysis to dissect the molecular heterogeneities in HSCs. We profile the transcriptomes of 180 highly purified HSCs (Bcl11a+/+ and Bcl11a−/−). Detailed analysis of the RNA-seq data identifies cell cycle activity as the major source of transcriptomic variation in the HSC compartment, which allows reconstruction of HSC cell cycle progression in silico. Single-cell RNA-seq profiling of Bcl11a−/− HSCs reveals abnormal proliferative phenotypes. Analysis of lineage gene expression suggests that the Bcl11a−/− HSCs are constituted of two distinct myeloerythroid-restricted subpopulations. Remarkably, similar myeloid-restricted cells could also be detected in the wild-type HSC compartment, suggesting selective elimination of lymphoid-competent HSCs after Bcl11a deletion. These defects are experimentally validated in serial transplantation experiments where Bcl11a−/− HSCs are myeloerythroid-restricted and defective in self-renewal.ConclusionsOur study demonstrates the power of single-cell transcriptomics in dissecting cellular process and lineage heterogeneities in stem cell compartments, and further reveals the molecular and cellular defects in the Bcl11a-deficient HSC compartment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0739-5) contains supplementary material, which is available to authorized users.
Highlights
Hematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages
We have previously reported that the C2H2 zinc finger transcription factor B cell CLL/lymphoma 11A (Bcl11a) is essential for both fetal and adult lymphoid development [9, 10]
(See figure on previous page.) Fig. 4 Bcl11a-deficient HSCs showed significant proliferative changes in the HSC compartment. a Distribution of Bcl11a−/− HSCs and cell cycle reconstructed-Bcl11a+/+ HSC clusters (C1–C5) in a principal component analysis (PCA) loading plot of the first two principal components. b Boxplots comparing the transcriptional activity of G0/G1 stage (C1 and C2) and S/G2/M stage (C3 and C4) HSCs in the Bcl11a+/+ and Bcl11a−/− datasets, estimated by the total number of read counts normalized by External RNA Controls Consortium (ERCC) size factor per cell. c Violin plots of gene expression of selected cyclin genes, progenitor markers and cell cycle stage-associated genes in Bcl11a+/+ and Bcl11a−/− HSCs
Summary
Hematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages. Recent studies have identified significant cellular heterogeneities in the HSC compartment with subsets of HSCs displaying lineage bias. We previously discovered that the transcription factor Bcl11a has critical functions in the lymphoid development of the HSC compartment. We have previously reported that the C2H2 zinc finger transcription factor B cell CLL/lymphoma 11A (Bcl11a) is essential for both fetal and adult lymphoid development [9, 10]. It is possible that the lymphoid defect in Bcl11a-deficient HSCs is dependent on its functions in the regulation of lineage-priming heterogeneities in the HSC compartment. This technical obstacle has recently been overcome by the advance of RNA-seq technology [16, 17], which allows genomewide transcriptome analysis to be conducted at the single-cell level
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