CD34⁺CD133⁺CD90⁺ hematopoietic stem cells (HSCs) are responsible for long-term multilineage hematopoiesis, and the high frequency of gene-modified HSCs is crucial for the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the ex vivo culture and gene manipulation steps of HSPC graft preparation significantly reduce the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the manipulation. In this study, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34⁺CD133⁺CD90⁺ HSCs over other subpopulations of adult HSPCs in ex vivo culture. The preferential expansion enriches the HSCs in ex vivo culture, enhances the adhesion, and results in a sixfold increase in the long-term engraftment in NSG mice. Further, the culture-enriched HSCs are more responsive to gene modification by lentiviral transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-fold in vivo. The yield of gene-modified HSCs obtained by the culture enrichment is similar to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by developing and demonstrating a facile HSPC culture condition that increases the frequency of gene-modified cells in vivo. This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

中文翻译：

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人CD34+CD133+CD90+造血干细胞的优先扩增提高基因治疗的基因修饰细胞频率

CD34 ⁺ CD133 ⁺ CD90 ⁺造血干细胞 (HSCs) 负责长期多系造血，基因修饰的 HSCs 的高频率对于造血干细胞和祖细胞 (HSPC) 基因治疗的成功至关重要。然而，HSPC 移植物制备的离体培养和基因操作步骤显着降低了 HSC 的频率，因此需要大剂量的 HSPC 和试剂进行操作。在这项研究中，我们确定了一种小分子、白藜芦醇、UM729 和 SR1 的组合，它们在离体中优先扩展 CD34 + ^CD133 + ^CD90 + ^HSCs而不是成人 HSPCs 的其他亚群文化。优先扩增丰富了离体培养中的 HSC，增强了粘附性，并导致 NSG 小鼠的长期植入增加了六倍。此外，富含培养的 HSC 对慢病毒转导和基因编辑的基因修饰反应更灵敏，使基因修饰的 HSC在体内的频率提高了 10 倍。通过培养富集获得的基因修饰的HSCs的产量与HSCs的分选纯化相似并且优于Cyclosporin-H处理。我们的研究通过开发和证明一种简便的 HSPC 培养条件来解决 HSPC 移植物中基因修饰的 HSC 频率低的关键挑战，该条件可增加体内基因修饰细胞的频率. 该策略将改善 HSPC 基因治疗的结果，并简化基因操作过程。