Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Sweden.
Division of Molecular Hematology, Lund Stem Cell Center, Lund University, Sweden.
Haematologica. 2020 Nov 1;105(11):2561-2571. doi: 10.3324/haematol.2019.234542.
Massive expansion of erythroid progenitor cells is essential for surviving anemic stress. Research towards understanding this critical process, referred to as stress-erythropoiesis, has been hampered due to lack of specific marker-combinations enabling analysis of the distinct stress-progenitor cells capable of providing radioprotection and enhanced red blood cell production. Here we present a method for precise identification and in vivo validation of progenitor cells contributing to both steady-state and stress-erythropoiesis, enabling for the first time in-depth molecular characterization of these cells. Differential expression of surface markers CD150, CD9 and Sca1 defines a hierarchy of splenic stress-progenitors during irradiation-induced stress recovery in mice, and provides high-purity isolation of the functional stress-BFU-Es with a 100-fold improved enrichment compared to state-of-the-art. By transplanting purified stress-progenitors expressing the fluorescent protein Kusabira Orange, we determined their kinetics in vivo and demonstrated that CD150+CD9+Sca1- stress-BFU-Es provide a massive but transient radioprotective erythroid wave, followed by multi-lineage reconstitution from CD150+CD9+Sca1+ multi-potent stem/progenitor cells. Whole genome transcriptional analysis revealed that stress-BFU-Es express gene signatures more associated with erythropoiesis and proliferation compared to steady-state BFU-Es, and are BMP-responsive. Evaluation of chromatin accessibility through ATAC sequencing reveals enhanced and differential accessibility to binding sites of the chromatin-looping transcription factor CTCF in stress-BFU-Es compared to steady-state BFU-Es. Our findings offer molecular insight to the unique capacity of stress-BFU-Es to rapidly form erythroid cells in response to anemia and constitute an important step towards identifying novel erythropoiesis stimulating agents.
红系祖细胞的大量扩增对于应对贫血应激至关重要。由于缺乏能够分析具有辐射保护和增强红细胞生成能力的特定应激祖细胞的特异性标志物组合，因此对这一关键过程（称为应激性红细胞生成）的研究受到了阻碍。在这里，我们提出了一种用于精确鉴定和体内验证有助于稳态和应激性红细胞生成的祖细胞的方法，从而首次能够对这些细胞进行深入的分子特征分析。在小鼠辐射诱导的应激恢复过程中，表面标志物 CD150、CD9 和 Sca1 的差异表达定义了脾脏应激祖细胞的层次结构，并且与最先进的方法相比，提供了功能应激-BFU-E 的高纯度分离，富集度提高了 100 倍。通过移植表达荧光蛋白 Kusabira Orange 的纯化应激祖细胞，我们确定了它们在体内的动力学，并证明 CD150+CD9+Sca1-应激-BFU-Es 提供了大量但短暂的辐射保护性红细胞波，随后由 CD150+CD9+Sca1+多潜能干细胞/祖细胞进行多谱系重建。全基因组转录分析显示，与稳态 BFU-Es 相比，应激-BFU-Es 表达的基因特征更与红细胞生成和增殖相关，并且对 BMP 有反应。通过 ATAC 测序评估染色质可及性揭示了应激-BFU-Es 中染色质环转录因子 CTCF 的结合位点的增强和差异可及性。我们的研究结果为应激-BFU-Es 快速形成红细胞以应对贫血的独特能力提供了分子见解，并为鉴定新的红细胞生成刺激剂迈出了重要一步。
