Epidermal Growth Factor Rejuvenates Aged Hematopoietic Stem Cells

Hematopoietic aging is associated with decreased hematopoietic stem cell (HSC) self-renewal capacity and increased risk for myelodysplasia and leukemia. Deficient DNA repair contributes to the decline in HSC self-renewal capacity during aging and it remains unclear whether extrinsic signals can rejuvenate aged HSCs. Here, we demonstrate that augmentation of non-homologous end-joining (NHEJ) DNA repair in aged HSCs via treatment with epidermal growth factor (EGF) rejuvenates HSC function. Seven day culture of BM CD34-ckit+sca-1+lin- (34-KSL) HSCs from aged C57BL/6 mice with EGF suppressed myeloid skewing and increased production of multipotent CFU-granulocyte, erythroid, monocyte and megakaryocyte (CFU-GEMM) colonies. Aged, EGF-treated HSCs displayed increased donor multilineage engraftment in primary competitively transplanted mice and in secondary mice compared to mice transplanted with aged, control HSCs. Donor cell engraftment within the bone marrow (BM) KSL and SLAM+KSL HSC population was > 2-fold increased in mice transplanted with aged, EGF-treated HSCs. Systemic administration of EGF to aged mice for 6 weeks also increased long term – HSC self-renewal capacity as measured by increased donor bone marrow (BM) competitive repopulation in primary and secondary transplanted mice. Conversely, deletion of EGFR in Scl/Tal1+ hematopoietic cells was associated with increased myeloid skewing and depletion of LT-HSCs in middle aged mice. Mechanistically, EGF treatment decreased DNA damage in aged HSCs through activation of DNA PK-cs, Artemis and NHEJ repair. Inhibition of DNA PK-cs blocked EGF-mediated restoration of multipotent differentiation and suppression of myeloid skewing in aged HSCs, suggesting that the restoration of hematopoietic potential in aged HSCs is dependent on EGF-mediated activation of DNA PK-cs. EGF treatment also converted the transcriptome of aged HSCs from enrichment for genes involved in cell death and survival to genes involved in HSC generation and identity. These data suggest that extrinsic activation of EGFR signaling can restore key functional capacities in aged HSCs. Overall design: For single-cell RNA sequencing (scRNA-seq), BM CD34-KSL cells were sorted from mice after subcutaneous injection of 1 µg/g EGF or saline in 200 µL daily for 3 days. RNA was extracted using the Chromium Single Cell 3' V2 Reagents Kits. The libraries were sequenced on the NovaSeq 6000 platform. Sequence reads were aligned and analyzed using the Cell Ranger Count pipeline (v7.0.1) (10X Genomics) to generate cell-associated gene expression matrices. The expression data was then normalized via Scran deconvolution36 and differential expression among samples was measured with DESeq 2 algorithm in Partek Flow. UMAP plots were generated in R v4.2.2 using the Seurat v4.2.1 package following dimensionality reduction using the first ten principal components while density plots were generated using ggplot2 v3.4.0 and the stat_density_2d function. Statistical filters of p < 0.05, FDR < 0.05, and fold change > 1.5X were applied prior to Ingenuity Pathway Analysis