诺扬生物

WAKO:小分子抑制剂：解锁iPSC的新潜能（后篇）

诺扬生物

2024-06-13

诱导多能干细胞（iPSC）凭借其遗传可塑性与近乎无限的扩增能力，为患者特异性疾病建模、高通量药物研发、毒理学研究以及再生医药带来了史无前例的机会。尽管如此，iPSC的临床转化依然面临着规模化与生产上的巨大挑战，因此急需发展大规模且经济效益高的细胞培养法。富士胶片新推出的Complete CEPT Cokctail与GMP规格Y-27632 Rock抑制剂能够支持iPSC的上下游产线，为iPS细胞疗法研发的各个阶段提供综合解决方案。

小分子抑制剂⸺推动干细胞技术

想要跨越前临床与临床环境之间的转化鸿沟，需要我们以远超现今技术所能允许的量生产iPSC。另外，成功的培养iPSC高度依赖个人技术水平，既难以传授，也难以囊括在自动化生产流程中。

使用ROCK抑制剂改善iPSC存活率

抑制Rho激酶（ROCK）能够解决部分上述问题。Rho激酶是一种丝氨酸/苏氨酸激酶，能够调节细胞骨架的收缩与重组。ROCK活性与一系列细胞通路相关，包括细胞凋亡、迁移、增殖以及分化²²，而抑制它能够促进干细胞的生存²⁴。Y-27632是目前有效且具选择性的ROCK抑制剂²⁵。在2007年的一份具历史意义的研究中首次展现了Y-27632对人ESC的生存、分化潜能以及分化能力的正面效果（图3）²⁶。后续研究总结了Y-37632在iPSC细胞的复苏与传代中的应用²² ，因此，在单细胞解离前向iPSC培养基中添加10 μM Y-27632成为了iPSC领域的标准操作。

Y-27632通过抑制细胞收缩以及随后的细胞凋亡和失巢凋亡介导的细胞死亡提高细胞的存活率²⁷。Y-27632添加剂能够提高单个细胞的存活率，从而提升接种及克隆效率，支持解离细胞或在无饲养层环境传代的细胞的未分化生长，促进胚体形成，改善冻存与分化操作²⁸ ，这对iPSC培养而言可谓无价之宝。

图3. Y-27632改善了解离后人ESCs的生存

A：Y-37632、caspase抑制剂，神经营养因子混合物（BDNF/NT-3/NT-4）与凋亡细胞的百分比。

B：培养解离细胞2，4，6天后细胞的数量。（From Watanabe et al. 2007, Nat Biotechnol)。

CEPT：崭露头角

尽管优势显著，但Y-27632调控的ROCK抑制并不能从影响细胞稳态、结构与功能的应激机制中保护iPSCs¹⁷ 。为了克服这些难题，美国国家转化科学促进中心（NCATS）的研究者们通过高通量筛选超过15000种小分子后研发了一种4组分混合物¹⁷。CEPT包括Chroman 1²⁹⸺一种比Y-27632更强力也更具选择性的ROCK抑制剂，泛caspase抑制剂Emricasan，整合应激反应（ISR）抑制剂Trans-ISRIB，以及多胺溶液，能够协同改善在基础研究、转化研究以及后续应用中iPSC的生存状况。

现有研究^17，30-32（图4）显示，CEPT混合物能够快速起效，且具有独特的细胞保护性质，能够克服干细胞工艺流程中固有的应激源，这些应激源往往会导致DNA损伤并造成细胞死亡。CEPT添加剂能够支持解离后细胞的结构与功能完整性，因此尤其适用于需求单细胞分离的应用，还能在长期单细胞传代与冻存中维持iPSC细胞系的多能性、染色体组型与分化能力。此外，CEPT还能提升冻存分化细胞的生存率，以及增加衍生细胞的功能性。最后，CEPT与Y-27632以及其他市售试剂相比（CloneR，RevitaCell，SMC4）选择性更强，效果更好，只需低成本就能获得更好的细胞保护效果。

图4. CEPT改善了人ESCs及iPSCs的生存与其克隆的生长，并提高了细胞冻存与功能表现

A：小分子混合物对hESC集落数量与规模的影响。

B：细胞凋亡。

C：冻存hESCs细胞的生存状况。

D：iPSC衍生细胞。

E：CEPT促进了hESCs中的单细胞克隆。

F：iPSC衍生心肌细胞解冻5天后的电生理特征。(引用至 Chen et al. 2021, Nat Methods)

展望⸺iPSC的瓶颈与下一步

人iPSCs能够在体外环境下再现受体表现型的能力为生物医药研究带来了变革，并应用在患者定制疾病建模、高通量药物筛查、安全药理学以及再生医药等领域。尽管iPSC提供了前所未有的机遇，想要全面发挥它的潜能还需走很长的路。iPSC在临床转化上的困难之一就是成本，最新的评估³⁴ 指出一个研究级别iPSC细胞系的生产、表征验证以及储存需要花费1万到2万5千美元，而能供移植使用的GMP规格iPSC细胞疗法的费用则更为高昂，平均每位病人约为80万美元。自体疗法需要严格的操作与质量控制，费用会更加昂贵。尽管自体疗法可能是优势的iPSC应用之一，但它的价格可能会限制它的发展，使大多数公司选择生产研发能够用于特定疾病病人的“现货型”异体iPSCs细胞系。

除了工艺的复杂性外，在不考虑质量或其他考量（如致瘤性、异质性和免疫原性⁵的前提下，生产iPSC细胞的成本主要来自其低生存率。这限制了我们高效地把生产规模扩大至临床应用水平。另一大阻碍是细胞培养技术缺乏统一标准，但大力投资iPSC重编程及扩增的优化与自动化方面已成为新的行业趋势。

Y-27632的特性及其对细胞生存和细胞活力的影响标志着干细胞研究的转折点。十多年来，Y-27632添加剂已成为干细胞研究的标准操作，所以关于其在改善细胞生存的优势与机制方面积累了丰富的知识。为了iPSC应用的高效发展，干细胞药企越来越多地转向使用Y-27632作为细胞疗法的上游原料，富士胶片和光纯药株式会社新推出的GMP规格Y-27632能够支持长期，cGMP级别的iPSC规模化培养以及下游分化。

在需要单细胞解离的研究应用中，全新的CEPT混合物则能够提供更多的优势。它在细胞保护方面的能力已经在多个领域得到应用，包括自动化iPSC培养³⁰、胚状体优化与类器官模型³³、定向神经分化^31，32 、胎盘发育及功能研究³⁵ 。更多研究也在不断发掘它在转化研究中的可用性。

CEPT优越的能力与目标特异性为iPSC高效培养提供了强有力的支持，为疾病模型、新药研发、组织工程及再生医药带来了广泛的影响。富士胶片和光纯药新研发的CEPT混合物由四种成分组成，能够提供优越的细胞保护效果以安全高效地进行iPSC重编程、长期细胞培养、单细胞克隆以及基因编辑、胚状体以及类器官形成、细胞冻存以及细胞库建立。

富士胶片集团一直致力于能够促进生物医药研究与应用的各种创新，新推出的iPSC上下游产品研发的综合解决方案专为推动iPSC技术从前临床阶段到商业生产而设计，有助于发挥iPSC在介入治疗以及提供治疗方案的临床潜能。

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