Lin Shi-Lung, Ying Shao-Yao
Division of Regenerative Medicine, WJWU & LYNN Institute for Stem Cell Research, Santa Fe Springs, CA, USA.
Department of Integrative Anatomical Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
Methods Mol Biol. 2018;1733:265-282. doi: 10.1007/978-1-4939-7601-0_22.
Today's researchers generating induced pluripotent stem cells (iPS cells or iPSCs) usually consider their pluripotency rather than potential tumorigenicity. Oncogenic factors such as c-Myc and Klf4 are frequently used to boost the survival and proliferative rates of iPSCs, creating an inevitable problem of tumorigenicity that hinders the therapeutic usefulness of these iPSCs. To prevent stem cell tumorigenicity, we have examined mechanisms by which the cell cycle genes are regulated in embryonic stem cells (ESCs). Naturally, ESCs possess two unique stemness properties: pluripotent differentiation into almost all cell types and unlimited self-renewal without the risk of tumor formation. These two features are also important for the use of ESCs or iPSCs in therapy. Currently, despite overwhelming reports describing iPSC pluripotency, there is no report of any tumor prevention mechanism in either ESCs or iPSCs. To this, our studies have revealed for the first time that an ESC-specific microRNA (miRNA), miR-302, regulates human iPSC tumorigenicity through cosuppression of both cyclin E-CDK2 and cyclin D-CDK4/6 cell cycle pathways during G1-S phase transition. Moreover, miR-302 also silences BMI-1, a cancer stem cell gene marker, to promote the expression of two senescence-associated tumor suppressor genes, p16Ink4a and p14/p19Arf. Together, the combinatory effects of inhibiting G1-S cell cycle transition and increasing p16/p14(p19) expression result in an attenuated cell cycle rate similar to that of 2-to-8-cell-stage embryonic cells in early zygotes (20-24 h/cycle), which is however slower than the fast proliferation rate of iPSCs induced by the four defined factors Oct4-Sox2-Klf4-c-Myc (12-16 h/cycle). These findings provide a means to control iPSC tumorigenicity and improve the safety of iPSCs for the therapeutic use. In this chapter, we review the mechanism underlying miR-302-mediated tumor suppression and then demonstrate how to apply this mechanism to generate tumor-free iPSCs. The same strategy may also be used to prevent ESC tumorigenicity.
如今，致力于诱导多能干细胞（iPS细胞或iPSCs）研究的人员通常关注的是这些细胞的多能性，而非其潜在的致瘤性。诸如c-Myc和Klf4等致癌因子常被用于提高iPSCs的存活率和增殖率，这就不可避免地产生了致瘤性问题，进而阻碍了这些iPSCs在治疗中的应用。为了防止干细胞致瘤性，我们研究了胚胎干细胞（ESCs）中细胞周期基因的调控机制。自然状态下，ESCs具有两种独特的干性特性：能多能分化为几乎所有细胞类型，以及具有无限自我更新能力且无肿瘤形成风险。这两个特性对于ESCs或iPSCs在治疗中的应用也很重要。目前，尽管有大量关于iPSC多能性的报道，但尚无关于ESCs或iPSCs中任何肿瘤预防机制的报道。对此，我们的研究首次揭示，一种ESCs特异性微小RNA（miRNA），即miR-302，在G1-S期转换过程中通过共抑制细胞周期蛋白E-CDK2和细胞周期蛋白D-CDK4/6细胞周期途径来调控人类iPSC的致瘤性。此外，miR-302还使癌症干细胞基因标志物BMI-1沉默，以促进两个衰老相关肿瘤抑制基因p16Ink4a和p14/p19Arf的表达。抑制G1-S细胞周期转换和增加p16/p14（p19）表达的联合效应共同导致细胞周期速率减缓，类似于早期受精卵中2至8细胞期胚胎细胞的速率（20 - 24小时/周期），然而这比由四种特定因子Oct4-Sox2-Klf4-c-Myc诱导的iPSCs的快速增殖速率（12 - 16小时/周期）要慢。这些发现提供了一种控制iPSC致瘤性的方法，并提高了iPSCs用于治疗的安全性。在本章中，我们将回顾miR-302介导的肿瘤抑制的潜在机制，然后展示如何应用这一机制来生成无肿瘤的iPSCs。同样的策略也可用于预防ESCs的致瘤性。
