Single Cell Analysis Elucidates the Maturation of Human Stem and Progenitor Cell Function from Fetal through Adult Hematopoiesis
Authors: Li Hojun , Ezike Jideofor , Afanassiev Anton , Greenstreet Laura , Zhang Stephen , Whangbo Jennifer S. , Butty Vincent , Moiso Enrico , Connelly Guinevere , Morris Vivian , Wang Dahai , Daley George Q. , Garg Salil , Chou Stella T. , Regev Aviv , Lummertz da Rocha Edroaldo , Schiebinger Geoffrey , Rowe R. Grant 
Published: November 2021

Abstract
Hematopoiesis continually replenishes the supply of circulating blood cells from embryonic development through the entirety of human lifespan. Although all hematopoietic lineages are produced throughout life, biases in lineage output occur at various stages, including lymphoid bias in childhood and myeloid bias in adulthood. Furthermore, many blood disorders demonstrate marked biases in age of onset, such as bone marrow failure disorders, clonal hematopoiesis of indeterminate potential, and numerous hematologic malignancies. A lack of insight into the normal physiologic changes occurring in the hematopoietic stem and progenitor cell (HSPC) compartments during development and maturation fundamentally limits our understanding of how age biased blood disorders arise. Two major unresolved questions are: (1) what changes in the molecular regulation of hematopoietic lineage commitment occur over the course of human life? And (2) are certain HSPC states present only during specific ages of life, and if so, do age-specific HSPCs have distinct biology?
To address these questions we performed single cell RNA sequencing (scRNAseq) on human HSPCs from first and second trimester fetal liver hematopoiesis, and bone marrow hematopoiesis spanning childhood into mature adulthood. In total, HSPC samples were obtained from 14 distinct human donors. Dimensionality reduction and marker gene analysis identified uncommitted hematopoietic stem cells (HSCs) and the developmental trajectories of each lineage emanating from multipotent HSCs. We then identified the genes activated upon commitment to each hematopoietic lineage during fetal, childhood, and mature adult hematopoiesis using the Population Balance Analysis and Stationary Optimal Transport algorithms, followed by Elastic Net gene regression. For each lineage we determined the putative transcription factor network that is consistently active in driving commitment to that lineage throughout life, but surprisingly also found the existence of adjunctive transcription factor networks that only drove lineage commitment at specific ages.
We next used unbiased clustering of scRNAseq data to identify 21 distinct subtypes in the HSPC compartment across human life. Using marker gene analysis and singleCellNet algorithm comparisons to an existing human adult bone marrow scRNAseq data set, we hierarchically ordered and annotated these HSPC subtypes ranging from uncommitted HSCs to lineage committed progenitor cells. We found that cellular distribution within the HSPC compartment amongst these subtypes varied markedly throughout human lifetime, with higher representation of HSCs in fetal life, predominance of lymphoid progenitors in childhood, and higher representation of myeloid progenitors in adulthood. Focusing on the distribution of cells among HSC subtypes over human life, we identified an HSC subtype exclusive to mid-gestation that was not present in early fetal or postnatal timepoints. This HSC subtype had a characteristic immunophenotype and was enriched for expression of early response transcription factors and mRNA decay factors. We functionally validated that this mid-gestation-specific HSC subtype was phenotypically unique using colony formation assays and xenotransplantation. Mid-gestation-specific HSCs were more clonogenic with a greater number of multi-lineage outcomes, and also demonstrated increased multilineage engraftment capacity compared to other HSC subtypes
Our findings reveal that the intrinsic biology of hematopoietic lineage commitment fundamentally changes over the course of the human lifetime, and define and validate age-specific HSPC subtypes. In particular, the biology of the mid-gestation-specific HSC we identified has potential applications for improving engraftment and multi-lineage reconstitution in hematopoietic cell transplantation.

Disclosures
Regev: Genentech: Current Employment; Celsius Therapeutics: Current equity holder in publicly-traded company, Other: Co-founder; Immunitas: Current equity holder in publicly-traded company; ThermoFisher Scientific: Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Neogene Therapeutics: Membership on an entity's Board of Directors or advisory committees; Asimov: Membership on an entity's Board of Directors or advisory committees.