Single-Cell Analysis of Human B Lymphoid and Neutrophil/Monocyte Lineage Restriction

Lifelong production of most types of mature blood cells is sustained by a small population of cells with extensive regenerative potential. However, the detailed steps that restrict multipotent or even bipotent human hematopoietic cells to any single lineage remain poorly understood. Those that segregate the human B-lymphoid and neutrophil/monocyte (NM) lineages are of particular interest as these appear to identify a stage that might control the different properties of human leukemias that display perturbed NM and/or B-cell programs. To undertake a refined analysis of this normal lineage restriction process in human cells, we first devised a culture system that permits it to be tracked clonally and efficiently (50%). Initial experiments showed this could be achieved using a combination of multiple stromal cell types and human growth factor-supplemented medium. To elucidate the intervening transitional steps we then used multiplexed flow cytometry to compare the progeny generated in this culture system over a 2-week time course from previously defined lymphoid progenitor-enriched (P-L), NM progenitor-enriched (P-NM) and less restricted P-mix cord blood (CB) subsets. The results suggested that gain of CD45RA (RA) expression and loss of CLEC12A (C) expression appeared to accompany the sequential restriction of early CD34+ progenitors first to cells with dual NM+B-lineage potential and then just to B-lineage potential.

Subsequent tracking of the lineage outputs of CD34+ RA-C- cells initially produced in larger numbers from unfractionated CD34+ CB cells either in vitro or in xenografted immunodeficient mice, confirmed the CD34+ RA-C- subset to be highly enriched in cells with dual NM+B potential. In contrast, co-generated CD45RA+ (RA+C-) and RA+CLEC12A+ (RA+C+) phenotypes displayed separate B- and NM lineage-restricted activity, respectively, as indicated by their largely exclusive clonal outputs of CD19+ pre-B and CD14+/CD15+ NM precursors. In agreement with these phenotypically established separate NM and B-lineage outputs, RA+C- cells were found to contain higher levels of B-lineage-associated gene transcripts (e.g., DNTT, CD79A, and EBF1), whereas RA+C+ cells contained higher levels of the NM transcription factor mRNAs encoded by SPI1 and CEBPA. In a further optimized stroma-free liquid culture system, the RA-C- cells could be shown to produce continuously RA+C- and RA+C+ progeny after another 3-4 days, and also RA-C- progeny which are not produced from more restricted RA+C- and RA+C+ cells, suggesting that the acquired expression of RA precedes the separation of NM and B-lineage potential that is then marked by the differential activation of C expression in RA+ cells.

To examine more precisely how the process of B+NM restriction to a single lineage might be related to successive cell cycles, we labeled RA-C- cells with carboxyfluorescein diacetate succinimidyl ester (CFSE) to enable the phenotypes and growth potential of the the progeny obtained after 6 days to be directly related to their prior division histories. This revealed extensive heterogeneity in the overall distribution of the initial progeny cell cycle times but with a clear segregation in the outputs of the faster and slower dividing cells. Notably, the faster dividers produced ultimately small M+B or uni-lineage clones whereas the initially slow dividers subsequently produced larger clones, 25% of which still contained CD34+ cells or N+M+B progeny.

Taken together, these findings identify new hallmark phenotypic changes that identify a critical step in the lineage restriction of primitive human hematopoietic cells with dual NM+B-lineage potential and a previously unknown association of this process with a shortening of their cell cycle transit time.