Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition.

Yumei Luo,Shu Wang,Xiuli An,Detu Zhu,Xuehu Xu,Xiaofang Sun

doi:10.1155/2016/3598542

Abstract

The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p, miR-199a-3p, and miR-214 in the microRNA gene cluster. Meanwhile, the expression levels of their targets related to regulation of hypoxia-stimulated cell migration, such as HIF1A, MET, and MAPK1, were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy.

Highlights

The past decade has seen the development and application of neural stem cells (NSCs) as a novel gene delivery vector for targeted cancer gene therapy from bench to bedside [1]
The epihiPSC line was characterized by Alkaline Phosphatase (AP) staining, immunostaining for the pluripotency marker Oct4, and embryoid body differentiation (Figure 1(a))
NSCs were derived from the above epi-human induced pluripotent stem cell (hiPSC) by a simple adherent monolayer culture method and characterized by immunostaining for nestin, an early-stage marker of NSCs (Figure 1(b))

Summary

Introduction

The past decade has seen the development and application of neural stem cells (NSCs) as a novel gene delivery vector for targeted cancer gene therapy from bench to bedside [1]. When brain tumors are present, NSCs are capable of migrating through the brain parenchyma to home in on the tumor foci, including the original site and distant metastatic sites [2]. This tumor-tropic behavior has been reported to be stimulated by chemokines released from hypoxic tumors via signaling pathways such as SDF-1/CXCR4 and HGF/c-Met [3].

Methods

Results

Conclusion