Mejía-Cruz Claudia Camila, Barreto-Durán Emilia, Pardo-Pérez María Alejandra, Jimenez María Camila, Rincón Julieth, Vanegas Karen, Rodríguez Jorge Luis, Jaramillo-Garcia Luis Fernando, Ulloa Juan Carlos, Díaz Rodolfo Martínez, Leal-García Efrain, Pérez-Núñez Rafael, Barreto Alfonso, Rodríguez-Pardo Viviana M
Immunobiology and Cell Biology Group, Department of Microbiology, Science Faculty, Pontificia Universidad Javeriana, Bogotá D.C., Colombia.
Department of Pathology, School of Medicine, Pontificia Universidad Javeriana, Hospital Universitario San Ignacio, Bogotá D.C., Colombia.
Int J Stem Cells. 2019 Mar 30;12(1):51-62. doi: 10.15283/ijsc18061.
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 mesenchymal 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.
Human bone marrow-mesenchymal stem cells, umbilical cord blood-hematopoietic stem cells and a non-tumoral endothelial cell line (CC2811, Lonza) 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 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.
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.
人类造血干细胞的特性受骨髓微环境的制约，在该微环境中它们与其他细胞群体相互作用，如间充质干细胞和内皮细胞；然而，对这种微环境的研究很复杂。本研究的目的是通过磁悬浮开发一种模拟人类造血干细胞微环境的三维培养系统。
使用人骨髓间充质干细胞、脐带血造血干细胞和一种非肿瘤性内皮细胞系（CC2811，Lonza公司），通过磁悬浮方法构建器官型多细胞球体。我们获得了平均球度指数大于0.6、平均体积为0.5立方毫米且聚集百分比大于70%的活结构。对器官型多细胞球体进行苏木精和伊红染色的组织学研究，并通过免疫组织化学评估波形蛋白表达，结果显示其内部结构有序，无固缩细胞，且波形蛋白表达较高。通过多能性测试评估了器官型多细胞球体培养后人造血干细胞的功能能力，结果表明，不含外源性Flt3L的三维结构在维持人造血干细胞多能性方面具有自主性。
我们利用正常人细胞构建了模拟人类造血干细胞微环境的器官型多细胞球体。这些结构是开发复杂类器官的原型，有助于进一步研究正常人干细胞的生物学特性及其在再生医学中的潜力。
