Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany; Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland;
Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany;
Blood. 2016 Sep 1;128(9):1181-92. doi: 10.1182/blood-2016-03-705590. Epub 2016 Jun 30.
The maintenance of hematopoietic stem cells (HSCs) during ex vivo culture is an important prerequisite for their therapeutic manipulation. However, despite intense research, culture conditions for robust maintenance of HSCs are still missing. Cultured HSCs are quickly lost, preventing their improved analysis and manipulation. Identification of novel factors supporting HSC ex vivo maintenance is therefore necessary. Coculture with the AFT024 stroma cell line is capable of maintaining HSCs ex vivo long-term, but the responsible molecular players remain unknown. Here, we use continuous long-term single-cell observation to identify the HSC behavioral signature under supportive or nonsupportive stroma cocultures. We report early HSC survival as a major characteristic of HSC-maintaining conditions. Behavioral screening after manipulation of candidate molecules revealed that the extracellular matrix protein dermatopontin (Dpt) is involved in HSC maintenance. DPT knockdown in supportive stroma impaired HSC survival, whereas ectopic expression of the Dpt gene or protein in nonsupportive conditions restored HSC survival. Supplementing defined stroma- and serum-free culture conditions with recombinant DPT protein improved HSC clonogenicity. These findings illustrate a previously uncharacterized role of Dpt in maintaining HSCs ex vivo.
维持造血干细胞（HSCs）在体外培养过程中的活力是对其进行治疗性操作的重要前提。然而，尽管进行了深入的研究，仍缺乏能够强力维持 HSCs 的培养条件。培养的 HSCs 会迅速丢失，从而阻碍了对其的进一步分析和操作。因此，有必要确定支持 HSCs 体外维持的新因子。与 AFT024 基质细胞系共培养能够长期维持 HSCs 的体外活力，但负责的分子机制仍不清楚。在这里，我们使用连续的长期单细胞观察，鉴定在支持性或非支持性基质共培养下 HSC 的行为特征。我们报告了早期 HSC 存活是维持 HSC 活力的主要特征。对候选分子进行操作后的行为筛选表明，细胞外基质蛋白真皮蛋白聚糖（Dpt）参与了 HSC 的维持。在支持性基质中敲低 DPT 会损害 HSC 的存活，而在非支持性条件下异位表达 Dpt 基因或蛋白则可以恢复 HSC 的存活。在定义明确的无基质和无血清培养条件下补充重组 DPT 蛋白可提高 HSC 的集落生成能力。这些发现说明了 Dpt 在维持 HSCs 体外活力方面的一个先前未被描述的作用。
