San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
National Research Council, Milan, Italy.
Stem Cells Transl Med. 2017 Feb;6(2):352-368. doi: 10.5966/sctm.2015-0414. Epub 2016 Sep 16.
Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains. Stem Cells Translational Medicine 2017;6:352-368.
异体来源的人胎儿源性神经干细胞（hfNSCs）目前正处于几种神经退行性疾病的临床评估阶段，其显示出良好的安全性，但在患者中移植后需要免疫抑制。源自患者特异性诱导多能干细胞（iPSCs）的神经前体细胞可能与用于治疗具有未满足医疗需求的遗传疾病的自体体外基因治疗应用相关。在这种情况下，获得具有可靠“神经干细胞特征”的 iPSC 衍生神经干细胞（NSCs）是必需的。在这里，我们通过对来自正常供体和患有黏脂贮积症（MLD）的患者的皮肤成纤维细胞进行重编程，生成了人 iPSC（hiPSC）克隆，MLD 是一种致命的神经退行性溶酶体贮积病，由芳基硫酸酯酶 A（ARSA）酶的遗传缺陷引起。我们将 hiPSCs 分化为 NSCs（hiPS-NSCs），它们在分子、表型和功能上与 hfNSCs 具有同一性，当我们在验证 hiPS-NSCs 的表型和预测其在脑内移植后的表现时，将其用作“金标准”进行平行比较。我们使用慢病毒载体有效地转导了 MLD hiPSCs，达到了超生理 ARSA 活性，并且在神经分化过程中进一步增加。将 hiPS-NSCs 脑内移植到新生和成年免疫缺陷的 MLD 小鼠中，可在整个中枢神经系统中稳定地恢复 ARSA 活性。重要的是，当使用过表达 ARSA 的细胞时，我们观察到硫酸脂的储存明显减少，而接受新生小鼠干预的小鼠比接受成年小鼠干预的小鼠具有明显的优势。因此，我们生成了一种可再生的 ARSA 过表达 iPSC 源性真正的 hNSCs 来源，与经过临床批准的 hfNSCs 相比具有改进的特征。患者特异性 ARSA 过表达 hiPS-NSCs 可用于自体体外基因治疗方案，以在 MLD 受影响的大脑中提供持久的酶供应。《干细胞转化医学》2017 年；6:352-368。
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