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Transcriptional programs of in vitro-expanded HSPCs with acute disruption of Lmo2 before and after co-culture with OP9-DLL1

ABSTRACT: Though Lmo2 is a well-established member of the hematopoietic stem/progenitor program, its function in normal T cell development has remained unclear and difficult to study. Lmo2’s quick downregulation in thymic-seeding progenitor cells makes it particularly challenging to precisely perturb for mechanistic analyses; however, a recent in vitro HSPC expansion method provides a platform for probing a greater number of progenitor cells during an extended window of time through the T cell DN stages. Consequently, we performed loss-of-function experiments in expanded HSPCs and analyzed subsequent gene expression with bulk RNA-seq and single cell RNA-seq to reveal Lmo2’s previously unknown role in early T cell development. Remarkably, premature loss of Lmo2 in expanded HSPCs results in the specific upregulation of the T cell program. Bulk RNA-seq at day 5 of OP9-Dll1 co-culture reveals that the DN1 (CD44+CD25-) population derived from expanded HSPCs with the Lmo2 knockout (KO) have higher expression of T cell-related genes, such as the TCR signaling genes Lat, Itk, Lck, Zap70, Cd3e, and Cd3g, and higher transcriptional activation of the T cell receptor (TCR)-C beta locus when compared to DN1 controls. These findings are supported by single cell RNA-seq (scRNA-seq), where Lmo2-KO cells have higher expression of T cell program members like Tcf7 and higher transcriptional activation of the (TCR)-C gamma locus by the day 3 timepoint. Interestingly, before entering a Notch environment, there is minimal difference between Lmo2 KO and their control counterparts by scRNA-seq. Furthermore, the d3 and d5 datasets do not strongly indicate that Lmo2 KO causes a downregulation of the stem/progenitor program. The scRNA-seq dataset also introduces the impact of Flt3L pre-treatment for 4 days of expansion (Flt3L-priming) before seeding onto OP9-DLL1 co-cultures. Differences due to Flt3L-priming can only be seen in a subpopulation of samples before entering a Notch signaling environment; by the day 3 timepoint, cells are virtually indistinguishable by priming, suggesting that developmental acceleration caused by Flt3L-priming is distinct from Lmo2-related mechanisms. Taken together, these datasets provide gene expression profiles of control-derived and Lmo2 KO-derived DN populations to present Lmo2 as an important controller of early T cell developmental kinetics.

ORGANISM(S): Mus musculus

PROVIDER: GSE287751 | GEO | 2026/01/01

REPOSITORIES: GEO

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Project description: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.

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Transcriptional programs of in vitro-expanded HSPCs with acute disruption of Lmo2 before and after co-culture with OP9-DLL1

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