Use single cell RNA sequencing to study the molecular siganature of primary human Megakaryocytic-erythroid progenitor

Megakaryocytic-Erythroid Progenitors (MEP) produce circulating red blood cells and platelets. Although much is known regarding megakaryocytic (Mk) and erythroid (E) maturation, detailed molecular mechanisms underlying the MEP fate decision have not been determined. Single cell RNA sequencing of highly enriched populations of primary human common myeloid progenitors (CMP), MEP, megakaryocyte progenitors (MKP) and erythroid progenitors (ERP), revealed that MEP have a distinct molecular signature with co-expression of genes otherwise expressed exclusively in CMP, MKP or ERP. Cell cycle genes are significantly differentially expressed between MEP, MKP, and ERP. We therefore tested the effects on MEP fate of genetic and pharmacologic modulation of cell cycle progression, and found that cell cycle activity mechanistically controls MEP fate decisions; cell cycle activation promotes E whereas cell cycle inhibition promotes Mk specification. The data obtained from healthy cells can now be applied to the mechanisms underlying benign and malignant disease states of Mk and E production. Overall design: To address the heterogeneity of the MEP enriched population, we performed single-cell mRNA sequencing (scRNA-seq) of FACS-enriched MEP, MKP and ERP, and CMP. Specifically, we tested whether MEP have an expression signature that is distinct from CMP, MKP and ERP. Single cells were captured and lysed using the Fluidigm C1 platform and sequenced to more accurately identify and profile the transcriptome of multi-lineage cells. On average, there were 504,984 aligned reads per cell, with an average of 5,028 genes expressed (FPKM>0.1) per cell. We first performed an analysis of the scRNA-seq data from sorted CMP, MEP, MKP and ERP populations from a single PBSC donor (donor-1, n=246 cells). Unsupervised analysis with the recently described ICGS software identified separate major gene expression clusters for each of the sorted populations along with subdivisions of the CMP, MEP, MKP and ERP. To confirm the major gene expression clusters associated with the four sorted populations, we performed scRNA-Seq analysis of cells from a different donor (donor-2, n=294 cells) sorted using a complementary gating strategy, but running 1 plate of MEPs and 1 plate of a mix of 55% MKP and 45% ERP.

149

Lu YC, Sanada C, Xavier-Ferrucio J, Wang L, Zhang PX, Grimes HL, Venkatasubramanian M, Chetal K, Aronow B, Salomonis N, Krause DS