Isolation, characterization and preclinical development of human glial-restricted progenitor cells for treatment of neurological disorders
Isolation, characterization and preclinical development of human glial-restricted progenitor cells for treatment of neurological disorders
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DOI：
10.2217/rme.10.24
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发表时间：
2010-05-01
影响因子：
2.7
通讯作者：
Campanelli, James T.
中科院分区：
文献类型：
--
作者：
Sandrock, Robert W.;Wheatley, Will;Campanelli, James T.
目的：胶质限制性祖细胞（GRP）是一种在体外和体内均可产生星形胶质细胞和少突胶质细胞的神经细胞群，作为治疗CNS脱髓鞘和神经退行性疾病的细胞治疗剂具有很大的前景。提供了用于临床环境中的人源GRP（hGRP;商品名Q-Cells（R））的制造和表征方案，其坚持严格的分离、繁殖、表征和储存标准。材料和方法：使用Miltenyi顺磁珠细胞分离技术，从胎龄17-24周的小鼠的胎儿尸体前脑组织中分离hGRP（通过其与A2 B5抗体的免疫反应性来定义）。GRP在确定的无异生物质培养基中生长6天。在收获时，使用免疫细胞化学技术表征hGRP。确定长期冷冻保存和储存条件以及冻融后的活力。hGRP的表型分化潜力通过植入到颤抖小鼠的CNS中的实验来确定。结果：在17-24周的胎龄期间，从50多个神经组织中分离出hGRP。细胞在体外无异生物质培养基中扩增至6天，表现出非常一致的免疫细胞化学特征。在体外生长6天后，未检测到纯化过程中使用的残留抗体。GRP可以以高达2400万个细胞/ml冷冻，并且在冻融后存活率超过70%。将解冻的hGRP移植到髓鞘发育不良颤抖小鼠模型的脑中，观察到分化为胶质细胞酸性蛋白阳性星形胶质细胞和髓鞘碱性蛋白阳性少突胶质细胞;未观察到人源性NeuN阳性神经元细胞，也未观察到异常细胞增殖。结论：我们证明，hGRP可以一致地获得，繁殖，冷冻保存和表征使用的协议，可以转移到一个良好的实验室规范/良好的生产规范设置的临床级hGRP细胞治疗剂的生产。功能数据表明，在这些条件下制造的细胞能够在髓鞘形成障碍的动物模型中分化成适当的细胞表型。
Aim: Glial-restricted progenitor cells (GRPs), a neural cell population that gives rise to astrocytes and oligodendrocytes both in vitro and in vivo, hold great promise as a cellular therapeutic for the treatment of demyelinating and neurodegenerative diseases of the CNS. The manufacturing and characterization protocols of human-derived GRPs (hGRPs; trade name Q-Cells (R)) for use in a clinical setting that adhere to rigorous standards for their isolation, propagation, characterization and storage are presented. Materials & methods: hGRPs, defined by their immunoreactivity with A2B5 antibodies, were isolated from fetal cadaver forebrain tissue of mice 17-24 weeks gestational age using Miltenyi paramagnetic bead cell separation technology. GRPs were grown in a defined xenobiotic-free medium for 6 days. At harvest, hGRPs were characterized using immunocytochemical techniques. Long-term cryopreservation and storage conditions, and viability upon freeze thaw were determined. The phenotypic differentiation potential of hGRPs was determined by implantation experiments into the CNS of shiverer mice. Results: hGRPs were isolated from over 50 neural tissues of either sex during gestational ages of 17-24 weeks. Cells expanded out to 6 days in vitro in a xenobiotic-free medium demonstrated very consistent immunocytochemical profiles. No residual antibody used in the purification process was detected after 6 days of growth in vitro. GRPs could be frozen at up to 24 million cells/ml and were over 70% viable upon freeze thaw. Thawed hGRPs transplanted into the brain of the dysmyelinated shiverer mouse model were observed to differentiate into both glial fibrillary acidic protein-positive astrocytes and myelin basic protein-positive oligodendrocytes; no human-derived NeuN-positive neuronal cells were observed and no abnormal cell proliferation was observed. Conclusion: We demonstrate that hGRPs can be consistently obtained, propagated, cryopreserved and characterized using protocols that can be transferred to a good laboratory practice/good manufacturing practice setting for the manufacture of clinical-grade hGRP cellular therapeutics. Functional data demonstrate that cells manufactured under these conditions are able to differentiate into appropriate cellular phenotypes in an animal model of dysmyelination.