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. 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

造血衰老与造血干细胞（hematopoietic stem cell, HSC）自我更新能力下降、骨髓增生异常综合征和白血病风险升高密切相关。DNA修复缺陷会加剧衰老过程中HSC自我更新能力的衰退，目前尚不清楚外源信号是否能够复壮衰老的HSC。本研究证实，通过表皮生长因子（epidermal growth factor, EGF）处理衰老HSC，增强非同源末端连接（non-homologous end-joining, NHEJ）DNA修复能力，可有效复壮HSC功能。将老年C57BL/6小鼠的骨髓（bone marrow, BM）CD34⁻cKit⁺Sca-1⁺Lin⁻（34-KSL）造血干细胞用EGF培养7天，可抑制髓系偏倚，并增加粒细胞-红细胞-单核细胞-巨核细胞多能集落形成单位（CFU-GEMM）的集落生成量。与移植老年对照HSC的小鼠相比，经EGF处理的老年HSC在原代竞争性移植小鼠和继代移植小鼠中，供体多系造血嵌合率均显著提升。移植经EGF处理的老年HSC的小鼠，其骨髓KSL及SLAM⁺KSL造血干细胞群体中的供体细胞嵌合率提升2倍以上。对老年小鼠全身性给予EGF共6周，同样可提升长期造血干细胞自我更新能力——该结果可通过原代和继代移植小鼠中供体骨髓竞争性造血重建能力的增强得以验证。反之，在Scl/Tal1⁺造血细胞中敲除EGFR，会导致中年小鼠出现髓系偏倚加剧以及长期造血干细胞（LT-HSC）耗竭。从机制上来说，EGF处理可通过激活DNA依赖蛋白激酶催化亚基（DNA-PKcs）、Artemis蛋白及NHEJ修复通路，降低衰老HSC的DNA损伤水平。抑制DNA-PKcs会阻断EGF介导的衰老HSC多向分化能力恢复以及髓系偏倚抑制，这表明衰老HSC的造血潜能恢复依赖于EGF介导的DNA-PKcs激活。EGF处理还可将衰老HSC的转录组从以细胞死亡与存活相关基因为富集特征，转变为以HSC生成及身份维持相关基因为富集特征。上述数据表明，外源激活EGFR信号通路可恢复衰老HSC的关键功能能力。本研究的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）流程如下：每日对小鼠皮下注射200 μL含1 μg/g EGF或生理盐水的溶液，连续注射3天后，分选骨髓CD34⁻KSL细胞。采用Chromium单细胞3’ V2试剂试剂盒提取RNA。构建的文库在NovaSeq 6000测序平台上进行测序。使用Cell Ranger Count分析流程（v7.0.1，10X Genomics）对测序reads进行比对与分析，生成细胞相关基因表达矩阵。随后通过Scran反卷积算法36对表达数据进行标准化，并在Partek Flow软件中使用DESeq2算法检测样本间的差异表达。使用R语言v4.2.2结合Seurat v4.2.1包，基于前10个主成分进行降维后生成UMAP图；密度图则通过ggplot2 v3.4.0的stat_density_2d函数生成。在进行Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）前，需预先设置统计学筛选阈值：p < 0.05、错误发现率（FDR）< 0.05且倍数变化>1.5倍。