Regeneration from human cord blood in SCID mice

为了检测人脐血细胞向视网膜神经细胞的转分化，我们使用了来自人脐带血的谱系阴性部分。首先，用淋巴提取法获得人脐血低密度组分。低密度细胞与生物素标记的抗CD3、CD19、CD14、CD15、CD16、CD56、CD11c抗体孵育，再与亲和素标记的磁珠孵育。用磁铁从低密度细胞中去除谱系细胞。残存细胞作为人脐血的谱系阴性细胞。用PKH26标记谱系阴性细胞，将其注入SCID小鼠视网膜下腔，用抗asialoGM1抗体去除其中的NK细胞。注射后两周，处死小鼠，取眼球。在组织学检查中，我们使用激光扫描共聚焦显微镜和实时RT-PCR检测到注射的人细胞在SCID小鼠的眼睛中。组织学上表达人巢蛋白、人MAP2、人神经元特异烯醇化酶、β-Ⅲ微管蛋白和视紫红质，实时RT-PCR法表达人视紫质基因。这些结果表明，人脐血细胞可以分化为视网膜神经细胞。

To examine trans-differentiation from human cord blood cells to retinal nerve cells, we used lineage negative fraction from human cord blood. First, low density fraction of human cord blood was obtained using lymphoprep. The low density cells were incubated with biotin-labeled anti-CD3, CD19, CD14, CD15, CD16, CD56, CD11c antibodies, followed by incubation with avidin-labeled magnetic beads. Lineage cells were deleted using a magnet from the low density cells. Residual cells were used as lineage negative cells of human cord blood. The lineage negative cells were labeled with PKH26 and they were injected into subretinal space of SCID mice, in which NK cells had been depleted using anti-asialoGM1 antibody. Two weeks after injection, we sacrificed the mice and obtained the eyes. In histological examination, we detected injected human cells in the eyes of the SCID mice, using a laser-scanning confocal microscope and real time RT-PCR. And the injected cells express human nestin, human MAP2, human neuron specific enolase,β-III tubulin and rhodopsin histologically and they expressed mRNA of human rhodopsin in real time RT-PCR. These results suggest that human cord blood cells can differentiate into retinal nerve cells.