HSCs with pulmonary signatures are mobilized during apheresis collections for transplantation. (A) PB stem cells of 8 healthy donors given granulocyte colony-stimulating factor (G-CSF) for mobilization were collected via apheresis and cryopreserved (sampling). Live/Lin⁻/CD34⁺ cells were flow sorted and encapsulated; 10× Chromium Single 3' v2 libraries were prepared, pooled, and sequenced (scRNA-seq). For donor demultiplexing via single nucleotide polymorphisms, bulkRNAseq was performed on Live/Lin⁺ cells. After Louvain clustering and annotation, phenotypic HSCs were subsetted from the mobilized pool and examined for their expression of canonical, lung, and BM HSC signature genes using UCell. (B) Basic demographics of the donor population. (C) Batch-corrected UMAP representation highlighting the HSC/MPP cluster (red), arrows indicate developmental trajectory into more committed progenies (erythroid, myeloid, lymphoid). (D) The total number of progenitor cells and number of HSCs per donor. Fraction of HSCs among all cells is given in red. (E) Pie graph showing the proportions of medullary (blue) and extramedullary (lung, red; other, gray) signatures in the HSC fraction of apheresis samples. (F) Bar graph showing the absolute numbers of HSCs across all donor that had a unique BM (blue) or lung (red) signature, cells that exhibited features of both BM and lung (violet), and cells that could not be assigned to either of these categories (gray). (G) Box and whisker plot representing the percentage of medullary and extramedullary signatures identified the HSC population. Dots represent the individual allogeneic donors. bulkRNAseq, bulk RNA sequencing.