Departamento de Neurologia e Neurocirurgia, Universiade Federal de São Paulo (UNIFESP) , São Paulo , Brazil ; Hospital Israelita Albert Einstein (HIAE), Instituto do Cérebro (InCe) , São Paulo , Brazil.
Hospital Israelita Albert Einstein (HIAE), Centro de Pesquisa Experimental (CPE) , São Paulo , Brazil ; Programa de Imunopatologia e Alergia da Faculdade de Medicina da USP (FMUSP) , São Paulo , Brazil.
Front Neurol. 2014 Jan 7;4:214. doi: 10.3389/fneur.2013.00214.
Glioblastomas are the most lethal primary brain tumor that frequently relapse or progress as focal masses after radiation, suggesting that a fraction of tumor cells are responsible for the tumor regrowth. The identification of a brain tumor cell subpopulation with potent tumorigenic activity supports the cancer stem cell hypothesis in solid tumors. The goal of this study is to determine a methodology for the establishment of primary human glioblastoma cell lines. Our aim is achieved by taking the following approaches: (i) the establishment of primary glioblastoma cell culture; (ii) isolation of neurospheres derived from glioblastoma primary cultures; (iii) selection of CD133 cells from neurospheres, (iv) formation of subspheres in the CD133-positive population, (v) study of the expression level of GFAP, CD133, Nestin, Nanog, CD34, Sox2, CD44, and CD90 markers on tumor subspheres. Hence, we described a successful method for isolation of CD133-positive cell population and establishment of glioblastoma neurospheres from this primary culture, which are more robust than the ones derived straight from the tumor. Pointed out that the neurospheres derived from glioblastoma primary culture showed 29% more cells expressing CD133 then the ones straight tumor-derived, denoting a higher concentration of CD133-positive cells in the neurospheres derived from glioblastoma primary culture. These CD133-positive fractions were able to further generate subspheres. The subspheres derived from glioblastoma primary culture presented a well-defined morphology while the ones derived from the fresh tumor were sparce and less robust. And the negative fraction of CD133 cells was unable to generate subspheres. The tumor subspheres expressed GFAP, CD133, Nestin, Nanog, CD44, and CD90. Also, the present study describes an optimization of neurospheres/subspheres isolation from glioblastoma primary culture by selection of CD133-positive adherent stem cell.
胶质母细胞瘤是最致命的原发性脑肿瘤，在放疗后常以局灶性肿块复发或进展，表明有一部分肿瘤细胞是肿瘤复发的原因。在实体瘤中，具有强大肿瘤形成活性的脑肿瘤细胞亚群的鉴定支持癌症干细胞假说。本研究的目的是确定建立原发性人胶质母细胞瘤细胞系的方法。我们的目标是通过以下方法实现：（i）建立原发性胶质母细胞瘤细胞培养；（ii）从胶质母细胞瘤原代培养物中分离神经球；（iii）从神经球中选择 CD133 细胞；（iv）在 CD133 阳性群体中形成亚球；（v）研究肿瘤亚球上 GFAP、CD133、Nestin、Nanog、CD34、Sox2、CD44 和 CD90 标志物的表达水平。因此，我们描述了一种从原代培养物中分离 CD133 阳性细胞群体并建立胶质母细胞瘤神经球的成功方法，该方法比直接从肿瘤中获得的神经球更健壮。指出，从胶质母细胞瘤原代培养物中获得的神经球中表达 CD133 的细胞比直接从肿瘤中获得的神经球多 29%，表明在从胶质母细胞瘤原代培养物中获得的神经球中 CD133 阳性细胞的浓度更高。这些 CD133 阳性细胞群能够进一步产生亚球。从胶质母细胞瘤原代培养物中获得的亚球具有明确的形态，而从新鲜肿瘤中获得的亚球稀疏且不够健壮。并且 CD133 阴性细胞群无法产生亚球。肿瘤亚球表达 GFAP、CD133、Nestin、Nanog、CD44 和 CD90。此外，本研究通过选择 CD133 阳性贴壁干细胞，描述了优化从胶质母细胞瘤原代培养物中分离神经球/亚球的方法。
