Using miRNA-mRNA Interaction Analysis to Link Biologically Relevant miRNAs to Stem Cell Identity Testing for Next-Generation Culturing Development
Using miRNA-mRNA Interaction Analysis to Link Biologically Relevant miRNAs to Stem Cell Identity Testing for Next-Generation Culturing Development
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DOI：
10.5966/sctm.2015-0154
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发表时间：
2016-06-01
影响因子：
6
通讯作者：
Vaes, Bart L. T.
中科院分区：
文献类型：
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作者：
Crabbe, Marian A. E.;Gijbels, Kristel;Vaes, Bart L. T.
干细胞的治疗益处已在多种疾病模型和临床试验中得到证实。强有力的质量保证对于培养程序的进步至关重要，以便在不影响治疗效力的情况下实现大规模细胞制造。microRNA（miRNAs或miRs）被证明是生物过程的主要调节因子，并且是潜在的理想质量标记。我们确定了在非临床多能成体祖细胞（MAPC）和间充质干细胞（MSC）培养条件下差异表达的miRNA标志物，这些标志物调节重要的干细胞特征，如增殖和分化。选择这些骨髓来源的干细胞类型是因为它们都发挥治疗功能，但具有不同的增殖和再生能力。为了确定细胞特异性标志物miRNA并评估其对干细胞质量的影响，对从三个共享供体分离的MAPC和MSC进行miRNA和mRNA分析。我们应用了一种基于整合RNA谱分析和生物学功能分析相结合的Intimity Pathway Analysis策略，以确定miRNA-mRNA相互作用对表型的影响。这导致了与细胞周期调控和发育相关的重要miRNA标志物的鉴定，最具特色的是MAPC标志物miR-204- 5 p和MSC标志物miR-335- 5 p，我们分别提供了其在分化和细胞周期调控中的功能的体外验证。重要的是，在无异种条件下以及在MAPC培养物的生物反应器分离和扩增期间维持标志物表达。总之，所鉴定的生物学相关miRNA标记物可用于在培养程序中实施变化时监测干细胞稳定性。
Therapeutic benefit of stem cells has been demonstrated in multiple disease models and clinical trials. Robust quality assurance is imperative to make advancements in culturing procedures to enable large-scale cell manufacturing without hampering therapeutic potency. MicroRNAs (miRNAs or miRs) are shown to be master regulators of biological processes and are potentially ideal quality markers. We determined miRNA markers differentially expressed under nonclinical multipotent adult progenitor cell (MAPC) and mesenchymal stem cell (MSC) culturing conditions that regulate important stem cell features, such as proliferation and differentiation. These bone marrow-derived stem cell types were selected because they both exert therapeutic functions, but have different proliferative and regenerative capacities. To determine cell-specific marker miRNAs and assess their effects on stem cell qualities, a miRNA and mRNA profiling was performed on MAPCs and MSCs isolated from three shared donors. We applied an Ingenuity Pathway Analysis-based strategy that combined an integrated RNA profile analysis and a biological function analysis to determine the effects of miRNA-mRNA interactions on phenotype. This resulted in the identification of important miRNA markers linked to cell-cycle regulation and development, the most distinctive being MAPC marker miR-204-5p and MSC marker miR-335-5p, for which we provide in vitro validation of its function in differentiation and cell cycle regulation, respectively. Importantly, marker expression is maintained under xeno-free conditions and during bioreactor isolation and expansion of MAPC cultures. In conclusion, the identified biologically relevant miRNA markers can be used to monitor stem cell stability when implementing variations in culturing procedures.