MiRNA-mediated control of exogenous OCT4 during mesenchymal-epithelial transition increases measles vector reprogramming efficiency

Ramya Rallabandi,Brenna Sharp,Conrad Cruz,Qi Wang,Alexis Locsin,Christopher B Driscoll,Ella Lee,Tim Nelson,Patricia Devaux

doi:10.1016/j.omtm.2021.11.012

Ramya Rallabandi, Brenna Sharp + Show 7 more

Open Access

https://doi.org/10.1016/j.omtm.2021.11.012

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Abstract

OCT4 is a key mediator of induced pluripotent stem cell (iPSC) reprogramming, but the mechanistic insights into the role of exogenous OCT4 and timelines that initiate pluripotency remain to be resolved. Here, using measles reprogramming vectors, we present microRNA (miRNA) targeting of exogenous OCT4 to shut down its expression during the mesenchymal to the epithelial transition phase of reprogramming. We showed that exogenous OCT4 is required only for the initiation of reprogramming and is dispensable for the maturation stage. However, the continuous expression of SOX2, KLF4, and c-MYC is necessary for the maturation stage of the iPSC. Additionally, we demonstrate a novel application of miRNA targeting in a viral vector to contextually control the vector/transgene, ultimately leading to an improved reprogramming efficiency. This novel approach could be applied to other systems for improving the efficiency of vector-induced processes.

Highlights

MV4FN expresses tricistron OSK instead of MeV hemagglutinin (H) and c-MYC in an additional transcription unit (ATU) after the H gene (Figure 1A, top genome). We modified this vector by isolating the OCT4 from a SOX2 and KLF4 bicistron (SK) in an individual ATU to make the MV(O)(SK)(M) vector (Figure 1A, middle genome)
All vectors showed comparable growth kinetics with the replication-competent MV(GFP) virus and replicated to maximum titers of greater than 106 by 48 h, confirming that vector propagation remains unaltered by the insertion of an additional ATU and the MTS375 in the 30UTR of OCT4 (Figure 1D)
There was no significant decrease in the expression of Vimentin (Figure S2), and this is mostly due to the presence of a high number of untransduced fibroblasts.[6]. These results indicate that MET occurs between days 9 and 15 and is cell maturity independent, as it occurs at a similar time frame in both neonatal human fibroblasts (NHFs) and adult human fibroblasts (AHFs)

Summary

Introduction

Reprogramming is a multi-dynamic molecular process involving conversion of somatic cells to induced pluripotent stem cells (iPSCs) via overexpression of four reprogramming factors (RFs): Oct[4], Sox[2], Klf[4], and c-Myc (OSKM)), which can be delivered using multiple vector systems.[1,2,3] Based on gene expression profiling, human somatic cell reprogramming is divided into two stages; initiation and maturation, linked by mesenchymal to epithelial transition (MET) phase, which is characterized by the activation of epithelial genes and repression of mesenchymal genes.[4,5,6] MET is orchestrated by exogenous RFsOSKM.[4,5,6,7,8] Oct[4], Sox[2], and Klf[4] are considered “pioneer factors” for inducing pluripotency.[8].

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