Investigation of the mesenchymal stem cell compartment by means of a lentiviral barcode library
Investigation of the mesenchymal stem cell compartment by means of a lentiviral barcode library
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通过慢病毒条形码文库研究间充质干细胞区室
DOI：
10.1134/s0006297916040076
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Drize NI
中科院分区：
文献类型：
--
作者：
Bigildeev AE;Cornils K;Aranyossy T;Sats NV;Petinati NA;Shipounova IN;Surin VL;Pshenichnikova OS;Riecken K;Fehse B;Drize NI
骨髓造血微环境是由间充质干细胞（mesenchymal stem cells，MSCs）增殖分化形成的。MSC隔室的研究少于造血干细胞隔室。为了表征MSC隔室的结构，有必要追踪不同间充质细胞的命运。为此，需要在单细胞水平上标记间充质祖细胞。在过去的10年里，已经开发出一种基于遗传"条形码"的正常和癌症干细胞个体标记方法。这种方法尚未应用于MSC。本研究的目的是评估使用这种条形码策略标记MSC及其后代成纤维细胞集落形成单位（CFU-Fs）的可能性。用具有由32 ± 3个简并核苷酸组成的条形码的慢病毒文库转导鼠长期骨髓培养物（LTBMC）的贴壁细胞层（CD4）。将感染的大肠杆菌悬浮，并获得CFU-F衍生的克隆。从每个单独的集落中分离DNA，并通过常规聚合酶链反应和桑格测序分析标记的CFU-F衍生集落中的条形码。在154个标记的菌落中鉴定了条形码。所有条形码似乎都是唯一的：没有两个不同的菌落带有相同的条形码。结果表明，骨髓间充质干细胞包括具有不同增殖潜能的CFU-Fs。MSC在间充质祖细胞的层级中比CFU-Fs更高，因此所呈现的数据表明MSC很少在LTBMC中增殖。本研究建立了一种稳定的间充质祖细胞个体标记和比较标记的方法。我们第一次证明了慢病毒的条形码文库是研究基质祖细胞的有效工具。
The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic “barcodes” has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F-derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.
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1.2
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作者：
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影响因子：
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作者：
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