Specific HLF expression in enriched human HSC populations. (A) HLF expression is enriched in cultured human HSC subsets. Differentially expressed genes from CD201⁺ vs CD201^mid/−7  and ITGA3⁺/CD201⁺/CD90⁺/CD133⁺/C34⁺/CD45RA⁻ (ITGA3⁺/CD201⁺) vs ITGA3⁻/CD201⁺/CD90⁺/CD133⁺/C34⁺/CD45RA⁻²¹  (ITGA3⁻/CD201⁺) after 7-day culture with UM171. Both transcriptomic surveys were integrated to identify consistently up- or downregulated genes. Ranking based on fold change between the most enriched (ITGA3⁺/CD201⁺) and most depleted (CD201⁻) HSC populations is summarized by a waterfall plot (left; log₂ transformed). Range of expression is provided for each gene in square brackets. Each population includes biological replicates represented along the x-axis. ITGA3⁻/CD201⁺ is abbreviated for the ITGA3⁻/CD201⁺/CD90⁺/CD133⁺/C34⁺/CD45RA⁻ population. (B) HLF expression is undetectable in mature blood leukocyte populations. A curated data set from Uhlen et al²² including between 4 and 6 biological replicates per population. (C) Single-cell transcriptomic overview of CB cell populations. CD34⁺ CB cells that were either thawed and noncultured (day 0) or UM171-expanded (day 7; 2 biological replicates for each condition; 15 921 total cells; fluorescence-activated cell sorting profiles are provided in supplemental Figure 2 green bars) were single-cell RNA sequenced (10X Chromium), integrated, and clustered using Seurat 3.⁵⁰ Eleven cell clusters were identified in the integrated data set: HSPCs, lymphoid-primed multipotent progenitors (LMPPs), multipotent progenitors (MPPs), granulomonocytic progenitors (GMPs), megakaryocyte erythroid mast cell progenitors (MEMPs), megakaryocytes (mega), eosinophil/basophils (eo/ba), mast cells (mast), erythroid lineage cells (erythroid), neutrophils (neutro), and monocytic/dendritic cells (mono/DCs). Dimensional reduction was calculated using SPRING.⁵¹ Combined representation of integrated data. Marker genes for each cluster are provided in supplemental Table 1. (D-E) HSPC-specific HLF expression in thawed noncultured CD34⁺ cells and after 7-day ex vivo expansion in the presence of UM171. HLF expression is shown in single-cell transcriptomes that are represented separately by treatment. Normalized expression data (z score computed separately by treatment after MAGIC imputation⁵²) is expressed in the color scale. (F) Comparison of HLF expression specificity vs selected HSC-associated genes and common HSC surface marker genes. Gene-wise z score distribution by treatment (fresh CD34⁺ and UM171 day 7) is represented as density for each cell community (same color code as in panel C). The mean z score for HLF in the HSPC cluster is provided as a dotted reference line for each treatment. (G-I) HLF expression is strongly enriched in HSC clusters in human bone marrow (BM). (G) Overview of cell clusters (Human Cell Atlas; 101 935 cells; integrated data from 8 donors; uniform manifold approximation and projection [UMAP] reduction; preprocessed data, clusters, and labels adopted from Hay et al³²). (H) HLF expression (z score normalized after MAGIC imputation). (I) Expression summary of selected HSC-associated genes in human BM (scaled expression averaged for each hematopoietic cell population and donor (n = 8) in the data set; row-normalized color scale). Nonhematopoietic cell populations were excluded from this representation. (J-L) HLF expression is restricted to the HSC/MPP cluster in hematopoietic human fetal liver (FL) cells. (J) Overview of cell communities (Human Cell Atlas; 113 063 cells; integrated data from 14 FLs across 4 developmental stages; UMAP reduction; preprocessed data, clusters, and labels adopted from Popescu et al³³). (K) HLF expression (z score normalized after MAGIC imputation). (L) Expression summary of HSC-associated genes in FL (scaled expression was averaged for each hematopoietic cell population and 4 gestation stages [7-8, 9-11, 12-14, and 15-17 postconception weeks]; row-normalized color scale). Nonhematopoietic cell populations were excluded. ILC, innate lymphoid cell; L/TL, early lymphoid/T lymphocyte; mac, macrophage; mono-mac, monocyte macrophage; neut-my, neutrophil myeloid; NK, natural killer; PBMC, peripheral blood mononuclear cell; pro, progenitor; TPM, transcripts per kilobase million.