Generation of Organotypic Multicellular Spheres by Magnetic Levitation: Model for the Study of Human Hematopoietic Stem Cells Microenvironment
Generation of Organotypic Multicellular Spheres by Magnetic Levitation: Model for the Study of Human Hematopoietic Stem Cells Microenvironment
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DOI：
10.15283/ijsc18061
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发表时间：
2019-03-01
影响因子：
2.3
通讯作者：
Rodriguez-Pardo, Viviana M.
中科院分区：
文献类型：
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作者：
Camila Mejia-Cruz, Claudia;Barreto-Duran, Emilia;Rodriguez-Pardo, Viviana M.
背景和目的：人类造血干细胞的特性受骨髓微环境的制约，在骨髓微环境中，它们与其他细胞群（如间充质干细胞和内皮细胞）相互作用；然而，对这种微环境的研究是复杂的。这项工作的目的是通过磁悬浮来开发一种模拟人类HSC微环境的3D培养系统。方法与结果：利用人骨髓间充质干细胞、脐带血造血干细胞和非肿瘤内皮细胞系（CC2811, Lonza (R)），采用磁悬浮法制备器官型多细胞球。我们获得了平均球形指数大于0.6，平均体积为0.5 mm(3)，聚集率大于70%的可行结构。用苏木精染色和伊红染色对多细胞球进行组织学研究，并用免疫组织化学方法对波形蛋白表达进行评估，结果表明其内部结构有组织，没有小细胞，波形蛋白高表达。通过多能性测试评估人造血干细胞在器官型多细胞球培养后的功能能力，结果表明，不含外源性Flt3L的3D结构在维持人造血干细胞多能性方面是自主的。结论：我们从正常人类细胞中培养出了器官型多细胞球，模拟了人类造血干细胞的微环境。这些结构是复杂类器官发展的原型，可以进一步研究正常人类干细胞的生物学及其在再生医学中的潜力。
Background and Objective: The characteristics of human hematopoietic stem cells are conditioned by the microenvironment of the bone marrow, where they interact with other cell populations, such as mcsenchymal stem cells and endothelial cells; however, the study of this microenvironment is complex. The objective of this work was to develop a 3D culture system by magnetic levitation that imitates the microenvironment of human HSC.Methods and Results: Human bone marrow-mesenchymal stem cells, umbilical cord blood hematopoietic stem cells and a non-tumoral endothelial cell line (CC2811, Lonza (R)) were used to develop organotypic multicellular spheres by the magnetic levitation method. We obtained viable structures with an average sphericity index greater than 0.6, an average volume of 0.5 mm(3) and a percentage of aggregation greater than 70%. Histological studies of the organotypic multicellular spheres used hematoxylin and eosin stains, and an evaluation of vimentin expression by means of immunohistochemistry demonstrated an organized internal structure without picnotic cells and a high expression of vimentin. The functional capacity of human hematopoietic stem cells after organotypic multicellular spheres culture was evaluated by multipotency tests, and it was demonstrated that 3D structures without exogenous Flt3L are autonomous in the maintenance of multipotency of human hematopoietic stem cells.Conclusions: We developed organotypic multicellular spheres from normal human cells that mimic the microenvironment of the human hematopoietic stem cells. These structures are the prototype for the development of complex organoids that allow the further study of the biology of normal human stem cells and their potential in regenerative medicine.