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Developmental biology - Reprogramming Cells to Pluripotency

A New way to turn skin cells into stem cells

For the first time skin cells are being converted into pluripotent stem cells using their own genes...


Our bodies consist of many different kinds of cells with a role to play in building and maintaining our body. But in 2012, two scientists: John Gurdon of the UK, and Shinya Yamanaka of Japan, came up with a winning recipe to induce adult skin cells to return to an early embryonic, and therefore pluripotent, state.

For their discovery, Gurdon and Yamanaka were awarded The Nobel Prize in Physiology or Medicine. The cells they manipulated are now referred to as "induced Pluripotent Stem Cells (iPSC)", and their process of cell reconversion called "reprogramming."

Since 2012, reprogramming skin cells has only been possible by introducing the critical 'Yamanka' genes, named after one of their codiscoverers, to artificially induce differentiated cells to once again become pluripotent.

But now, and for the first time, Professor Timo Otonkoski at the University of Helsinki and Professor Juha Kere at both Karolinska Institute and King's College London, have succeeded in reprogramming skin cells into pluripotent stem cells using only skin cell genes. They achieved this feat using the gene editing technology CRISPRa. Their method incorporates a blunted version of Cas9 'gene scissors' that doesn't cut DNA. Therefore, it activates gene expression without mutating the genome.
CRISPRa graph
a. Schematic representation of dCas9VPH structure.
b. Schematic of NSC reprogramming into iPSCs with dCas9VPH mediated OCT4 activation.

"CRISPR/Cas9 can be used to activate genes... an attractive possibility for cellular reprogramming because multiple genes can be targeted at the same time. Reprogramming based on activation of endogenous genes rather than overexpression of transgenes is also, theoretically, a physiological way of controlling cell fate and may result in more normal cells. In this study, we show that it is possible to engineer a CRISPR activator system that allows robust reprogramming of iPSC."

Timo Otonkoski PhD, Research Programs Unit, Molecular Neurology and Biomedicum Stem Cell Centre, Faculty of Medicine, University of Helsinki, Helsinki, Finland; and Children’s Hospital, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland.

"The technology may find practical use in bio banking and many other tissue technology applications, as it opens up new insights into the mechanisms controlling early embryonic gene activation," adds Jere Weltner, PhD student and first author of the article published in Nature Communications.

Abstract
CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming.

Authors: Jere Weltner, Diego Balboa, Shintaro Katayama, Maxim Bespalov, Kaarel Krjutškov, Eeva-Mari Jouhilahti, Ras Trokovic, Juha Kere and Timo Otonkoski.


Acknowledgements
We thank J. Saarimäki-Vire, S. Eurola, H. Grym, A. Laitinen, M. Salmela, Y. Novik, I. Fransson, and A. Damdimopoulos for technical assistance with the work and S. Vuoristo for comments on the manuscript. This work has been supported by the 3i Regeneration project (number 40395/13; a TEKES Large Strategic Research Opening), Jane and Aatos Erkko Foundation, Academy of Finland (No. 297466 and 312437, Center of Excellence in Stem Cell Metabolism), Sigrid Jusélius Foundation, Novo Nordisk Foundation, Instrumentarium Science Foundation, the Doctoral Program in Biomedicine at University of Helsinki, Knut and Alice Wallenberg Foundation (KAW2015.0096), SNIC through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) under project b2014069, Bioinformatics and Expression Analysis core facility (BEA) and The Mutation Analysis Core Facility (MAF) at the Karolinska University Hospital.

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Jul 10, 2018   Fetal Timeline   Maternal Timeline   News   News Archive




Colonies of CRISPRa reprogrammed "induced pluripotent stem cells (iPSC).
Image: Otonkoski Lab, University of Helsinki.


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