Enrichment of hematopoietic stem/progenitor cells in the zebrafish kidney
Enrichment of hematopoietic stem/progenitor cells in the zebrafish kidney
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DOI：
10.1038/s41598-019-50672-5
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发表时间：
2019-10-02
影响因子：
4.6
通讯作者：
Traver, David
中科院分区：
文献类型：
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作者：
Kobayashi, Isao;Kondo, Mao;Traver, David
造血干细胞（HSC）维持整个生命的血液系统，并用于血液疾病的治疗方法。高纯度HSC的前瞻性分离对于理解HSC调控的分子机制至关重要。斑马鱼具有许多独特的优点，是研究造血的理想遗传模型。然而，由于缺乏特异性HSC标记物，还不可能纯化成年斑马鱼中的HSC。在这里，我们展示了通过两种HSC相关转基因gata 2a：GFP和runx 1：mCherry的组合来富集斑马鱼HSC。在硬骨鱼的主要造血器官肾脏中检测到gata 2a：GFP和runx 1：mCherry的双阳性分数（gata 2a（+）runx 1（+））约为0.16%。转录组分析显示，gata 2a（+）runx 1（+）细胞显示出典型的HSC分子特征，包括gata 2b、gfi 1aa、runx 1 t1、pbx 1b和meis 1b的上调。移植试验表明，长期再增殖的HSC在gata 2a（+）runx 1（+）组分中高度富集。相反，集落形成试验表明，gata 2a-runx 1（+）细胞含有丰富的红系和/或髓系引发的祖细胞。因此，我们的纯化方法在斑马鱼肾脏中的HSC是有用的，以确定所需的调节HSC的自我更新和分化的分子线索。
Hematopoietic stem cells (HSCs) maintain the entire blood system throughout life and are utilized in therapeutic approaches for blood diseases. Prospective isolation of highly purified HSCs is crucial to understand the molecular mechanisms underlying regulation of HSCs. The zebrafish is an elegant genetic model for the study of hematopoiesis due to its many unique advantages. It has not yet been possible, however, to purify HSCs in adult zebrafish due to a lack of specific HSC markers. Here we show the enrichment of zebrafish HSCs by a combination of two HSC-related transgenes, gata2a: GFP and runx1:mCherry. The double-positive fraction of gata2a: GFP and runx1: mCherry (gata2a(+) runx1(+)) was detected at approximately 0.16% in the kidney, the main hematopoietic organ in teleosts. Transcriptome analysis revealed that gata2a(+) runx1(+) cells showed typical molecular signatures of HSCs, including upregulation of gata2b, gfi1aa, runx1t1, pbx1b, and meis1b. Transplantation assays demonstrated that long-term repopulating HSCs were highly enriched within the gata2a(+) runx1(+) fraction. In contrast, colony-forming assays showed that gata2a-runx1(+) cells abundantly contain erythroid-and/or myeloid-primed progenitors. Thus, our purification method of HSCs in the zebrafish kidney is useful to identify molecular cues needed to regulate self-renewal and differentiation of HSCs.