Lung-derived exosome uptake into and epigenetic modulation of marrow progenitor/stem and differentiated cells.
Lung-derived exosome uptake into and epigenetic modulation of marrow progenitor/stem and differentiated cells.
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DOI：
10.3402/jev.v4.26166
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发表时间：
2015
影响因子：
16
通讯作者：
Quesenberry PJ
中科院分区：
文献类型：
--
作者：
Aliotta JM;Pereira M;Sears EH;Dooner MS;Wen S;Goldberg LR;Quesenberry PJ
我们的小组先前已经证明，小鼠全骨髓细胞（WBM），内化肺衍生的细胞外囊泡（LDEV）在培养表达肺上皮细胞特异性基因长达12周。此外，移植有肺囊泡调节骨髓的致死性照射小鼠的肺具有比移植有未操作的WBM的小鼠多5倍的WBM衍生的II型肺细胞。这些发现表明，细胞外囊泡修饰可能是开发基于骨髓细胞的细胞疗法的重要考虑因素。进行当前研究以确定LDEV稳定修饰的特定骨髓细胞类型。用羧基荧光素N-琥珀酰亚胺酯（CFSE）标记的LDEV培养小鼠WBM衍生的干/祖细胞（Lin-/Sca-1+）和分化的红系细胞（Ter 119+）、粒细胞（Gr-1+）和B细胞（CD 19+）。通过流式细胞术分离LDEV+细胞（CFSE+）和LDEV−细胞（CFSE−），并通过荧光显微镜观察，通过RT-PCR分析或置于长期传代培养中。此外，将鼠Lin-/Sca-1+细胞与从大鼠分离的CFSE标记的LDEV一起培养，并使用表面活性剂的物种特异性引物对LDEV+和-细胞进行RT-PCR分析（大鼠/小鼠杂交共培养物）。干/祖细胞和所有分化的细胞类型研究了在培养物中内化LDEV，但不均匀。一组肺上皮细胞基因的表达在LDEV+细胞中高于LDEV −细胞，并且这些基因的表达升高在长期培养中持续存在。大鼠/小鼠杂交共培养显示，培养4周后，LDEV+ Lin-/Sca-1+细胞中仅表达小鼠特异性表面活性剂B和C，表明稳定的从头基因表达。LDEV可以被培养物中存在于骨髓中的分化的和更原始的细胞内化，并且可以在内化后数周内在这些细胞中诱导稳定的从头肺上皮细胞基因表达。基因表达代表靶骨髓细胞的转录激活。这些研究作为确定骨髓细胞类型的基础，这些骨髓细胞类型可用于损伤肺上皮表面的过程的基于细胞的治疗。
Our group has previously demonstrated that murine whole bone marrow cells (WBM) that internalize lung-derived extracellular vesicles (LDEVs) in culture express pulmonary epithelial cell–specific genes for up to 12 weeks. In addition, the lungs of lethally irradiated mice transplanted with lung vesicle–modulated marrow have 5 times more WBM-derived type II pneumocytes compared to mice transplanted with unmanipulated WBM. These findings indicate that extracellular vesicle modification may be an important consideration in the development of marrow cell–based cellular therapies. Current studies were performed to determine the specific marrow cell types that LDEV stably modify. Murine WBM-derived stem/progenitor cells (Lin-/Sca-1+) and differentiated erythroid cells (Ter119+), granulocytes (Gr-1+) and B cells (CD19+) were cultured with carboxyfluorescein N-succinimidyl ester (CFSE)-labelled LDEV. LDEV+ cells (CFSE+) and LDEV− cells (CFSE−) were separated by flow cytometry and visualized by fluorescence microscopy, analyzed by RT-PCR or placed into long-term secondary culture. In addition, murine Lin-/Sca-1+ cells were cultured with CFSE-labelled LDEV isolated from rats, and RT-PCR analysis was performed on LDEV+ and – cells using species-specific primers for surfactant (rat/mouse hybrid co-cultures). Stem/progenitor cells and all of the differentiated cell types studied internalized LDEV in culture, but heterogeneously. Expression of a panel of pulmonary epithelial cell genes was higher in LDEV+cells compared to LDEV − cells and elevated expression of these genes persisted in long-term culture. Rat/mouse hybrid co-cultures revealed only mouse-specific surfactant B and C expression in LDEV+ Lin-/Sca-1+cells after 4 weeks of culture, indicating stable de novo gene expression. LDEV can be internalized by differentiated and more primitive cells residing in the bone marrow in culture and can induce stable de novo pulmonary epithelial cell gene expression in these cells for several weeks after internalization. The gene expression represents a transcriptional activation of the target marrow cells. These studies serve as the basis for determining marrow cell types that can be used for cell-based therapies for processes that injure the pulmonary epithelial surfaces.