Molecular Neurobiology Laboratory and Department of Psychiatry and Program in Neuroscience, Harvard Stem Cell Institute, McLean Hospital/Harvard Medical School, Belmont, MA 02478, USA.
Proc Natl Acad Sci U S A. 2011 Jun 7;108(23):9703-8. doi: 10.1073/pnas.1016443108. Epub 2011 May 23.
During early development, midbrain dopaminergic (mDA) neuronal progenitors (NPs) arise from the ventral mesencephalic area by the combined actions of secreted factors and their downstream transcription factors. These mDA NPs proliferate, migrate to their final destinations, and develop into mature mDA neurons in the substantia nigra and the ventral tegmental area. Here, we show that such authentic mDA NPs can be efficiently isolated from differentiated ES cells (ESCs) using a FACS method combining two markers, Otx2 and Corin. Purified Otx2(+)Corin(+) cells coexpressed other mDA NP markers, including FoxA2, Lmx1b, and Glast. Using optimized culture conditions, these mDA NPs continuously proliferated up to 4 wk with almost 1,000-fold expansion without significant changes in their phenotype. Furthermore, upon differentiation, Otx2(+)Corin(+) cells efficiently generated mDA neurons, as evidenced by coexpression of mDA neuronal markers (e.g., TH, Pitx3, Nurr1, and Lmx1b) and physiological functions (e.g., efficient DA secretion and uptake). Notably, these mDA NPs differentiated into a relatively homogenous DA population with few serotonergic neurons. When transplanted into PD model animals, aphakia mice, and 6-OHDA-lesioned rats, mDA NPs differentiated into mDA neurons in vivo and generated well-integrated DA grafts, resulting in significant improvement in motor dysfunctions without tumor formation. Furthermore, grafted Otx2(+)Corin(+) cells exhibited significant migratory function in the host striatum, reaching >3.3 mm length in the entire striatum. We propose that functional and expandable mDA NPs can be efficiently isolated by this unique strategy and will serve as useful tools in regenerative medicine, bioassay, and drug screening.
在早期发育过程中，中脑多巴胺能（mDA）神经元祖细胞（NPs）通过分泌因子及其下游转录因子的共同作用，从腹侧中脑区产生。这些 mDA NPs 增殖、迁移到它们的最终目的地，并在黑质和腹侧被盖区发育成成熟的 mDA 神经元。在这里，我们使用一种结合了两个标记物 Otx2 和 Corin 的 FACS 方法，从分化的 ES 细胞（ESCs）中高效分离出这种真正的 mDA NPs。纯化的 Otx2(+)Corin(+)细胞共同表达其他 mDA NP 标记物，包括 FoxA2、Lmx1b 和 Glast。使用优化的培养条件，这些 mDA NPs 可以连续增殖长达 4 周，增殖倍数接近 1000 倍，而其表型没有明显变化。此外，在分化过程中，Otx2(+)Corin(+)细胞有效地产生 mDA 神经元，这表现为 mDA 神经元标记物（如 TH、Pitx3、Nurr1 和 Lmx1b）和生理功能（如高效的 DA 分泌和摄取）的共表达。值得注意的是，这些 mDA NPs 分化成相对同质的 DA 群体，只有少量的 5-羟色胺能神经元。当移植到 PD 模型动物、无晶状体小鼠和 6-OHDA 损伤大鼠时，mDA NPs 在体内分化为 mDA 神经元，并产生了整合良好的 DA 移植物，导致运动功能障碍显著改善而无肿瘤形成。此外，移植的 Otx2(+)Corin(+)细胞在宿主纹状体中表现出显著的迁移功能，在整个纹状体中达到超过 3.3 毫米的长度。我们提出，通过这种独特的策略可以有效地分离出功能性和可扩增的 mDA NPs，它们将成为再生医学、生物测定和药物筛选的有用工具。
