Transcriptional and epigenetic programs of in vitro-expanded HSPCs and their T cell progenitors progeny

Hematopoietic stem and progenitor cells (HSPCs) in the bone marrow are the ultimate sources of all hematopoietic lineage cells, including T cells. However, gene expression programs and chromatin dynamics that guide the bone marrow progenitor cells to enter the T-development programs are not fully understood due to limited cell numbers and population heterogeneity. By exploiting the in vitro HSPC expansion approach, which effectively expands HSPCs with high T cell potentials, we monitored the gene expression programs and chromatin accessibility changes underlying the transition from the bone marrow progenitor stages to early T cell development stages. Notably, expanded HSPCs displayed strikingly similar chromatin accessibility profiles with early-stage T cell progenitors, representing their shared hematopoietic chromatin landscapes. However, a select set of genomic regions and target genes were specifically regulated as cells first received the strong Notch signaling and engaged with T-development conditions. These events included a robust chromatin opening and transcriptional activation of the T cell receptor (TCR)-C beta locus. In addition, well-known stem and progenitor-associated transcription factors were sharply repressed, often concerted with broad chromatin accessibility losses at those loci. These gene regulation targets were not an artifact of in vitro expanded HSPC-derived pro-T cells. The progeny of expanded HSPCs and freshly isolated HSPCs share the same T-lineage developmental trajectory at the single-cell transcriptome level, and their gene expression programs were highly similar. However, expanded HSPC-derived pro-T cells showed temporal differences in early T-development speed and progressed through pre-commitment stages slowly. From cytokine and chemokine screening, we found that a brief Flt3L pre-treatment during the 4-5 days of the expansion period could moderately accelerate the T-development kinetics of expanded HSPCs. Thus, we compared the chromatin accessibility programs, H3K27ac and H3K27me3 histone marks, and gene expression programs upon Flt3L treatment. Although chromatin state and transcriptional features were mostly not altered by Flt3L treatment at the bulk population levels, scRNA-seq results showed that a set of activation and stress-response genes were upregulated upon Flt3L stimulation. However, Flt3L-primed HSPCs developed through a normal T cell pathway. Together, these datasets (1) provide comprehensive gene expression and chromatin accessibility profiles of expanded HSPCs and their progeny pro-T cells, (2) reveal molecular events accompanied by bone marrow progenitor cells transition to the T cell program, and (3) suggest a slight modification of the expansion protocol for the use of T cell biology studies by adding acute Flt3L treatment.