PDF(Pubmed)
Abstract：
Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.
摘要：
长期重建造血干细胞（LT-HSC）用于通过干细胞移植治疗血液疾病。LT-HSC的极低丰度及其在体外培养过程中的快速分化阻碍了其临床应用。以前使用基质饲养层的发展，定义的培养基鸡尾酒，生物工程使HSC在培养中得以扩展，但主要是短期的HSC和祖细胞群，以幼稚的LT-HSC为代价。这里,我们报告了一个生物工程LT-HSC维持生态位的创建，重建生理细胞外基质组织，使用柔软的I型胶原水凝胶驱动血管周围基质细胞（PerSC）中的巢蛋白表达。我们证明了Nestin,由支持HSC的骨髓基质细胞表达，是细胞保护的，通过调节新陈代谢，对于PerSC中HIF-1α的表达是重要的。当将CD34+veHSC添加到包含表达巢蛋白/HIF-1α的PerSC的生物工程生态位中时，LT-HSC数量保持正常克隆和体内重建潜力，没有媒体补充。我们提供了概念证明，我们的生物工程生态位可以支持CRISPR编辑的HSC的存活。LT-HSC离体的成功编辑可以对血液疾病的治疗具有潜在影响。
