3139 – TRANSCRIPTIONAL, PROTEIN-LEVEL AND FUNCTIONAL PROFILING OF HUMAN FETAL LIVER-DERIVED HEMATOPOIETIC STEM CELLS AT SINGLE CELL RESOLUTION

The complex and tightly regulated process of human hematopoietic development culminates in the production of hematopoietic stem cells (HSCs), which subsequently undergo expansion in the fetal liver (FL). The establishment of a high-resolution molecular signature of FL-HSCs would provide insights into HSC biology with potential utility in the purification and expansion of engraftable HSCs ex vivo and the generation of HSCs from pluripotent cell sources. To profile HSCs at this developmental stage, we performed CITE-Seq, a technique that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies. To connect expression profiles with functional engraftment, we have coupled this with transplantation assays in immunocompromised mice. In these studies, three populations of human FL cells were used: CD34- cells, CD34+ cells and CD34+ cells further enriched by expression of GPI-80, a marker tightly linked to engraftment potential, to acquire additional resolution of HSCs capable of long-term engraftment. These populations were stained with a panel of oligo-tagged antibodies, processed via the 10X platform, and sequenced (26.582 total cells). Simultaneous transplantation experiments of these three populations revealed superior engraftment potential of the CD34+GPI-80+ fraction, and thus enrichment for bona fide HSCs at the functional level. This coincided with enrichment for known HSC markers such as ITGA6 (CD49f), PROCR (EPCR), CD164, MLLT3, HLF and HMGA2 at the transcriptional level. As such, by profiling profiling \u003e7000 GPI-80+CD34+ cells, we have achieved unprecedented resolution of the engraftable HSC compartment within the FL. Studying the transcriptional profile in combination with protein level expression of key HSC-related genes, will allow for an accurate characterization of these cells and provide insights into the biology behind their remarkable engraftment potential.