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Gene-edited, stem cell reporter line could boost cardiac research
  • USA - English

There is an increasing need for stable stem cell lines that allow electrical or potential activities of the progeny heart muscle cells to be clearly and easily recorded. A study released today in STEM CELLS details the development of one such line, CRISPR-generated ArcLight-hiPSCs.


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STEM CELLS

Sep 30, 2019, 13:00 ET

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DURHAM, N.C., Sept. 30, 2019 /PRNewswire-PRWeb/ -- As human induced pluripotent stem cells (hiPSCs) move closer to becoming a possible mainstream therapy and an accepted model for studying the development and diseases of the human heart, there is an increasing need for stable stem cell lines that allow electrical or potential activities of the progeny heart muscle cells to be clearly and easily recorded. A study released today in STEM CELLS details the development of one such line, CRISPR-generated ArcLight-hiPSCs.

The UC Davis Health research team that developed the line reported that it incorporates a tracking process that overcomes several drawbacks inherent in other methods for recording how hiPSC-derived cardiomyocytes (hiPSC-CMs) function. This feature makes CRISPR-generated ArcLight-hiPSC a promising tool for studying these cells and utilizing them for drug testing.

Many researchers believe that hiPSC-CMs hold great potential for helping overcome heart disease, but a major challenge has been finding a suitable way to assess how well they function. The gold standard of cellular electrophysiology — patch-clamps — is too labor intense and invasive for assessing these cells, while non-invasive optical mapping with voltage-sensitive dyes such as FluoVolt could generate toxic metabolites that lead to phototoxicity and limit repeated long-term recordings.

That's why genetically encoded fluorescent voltage sensors (GEVIs) are gaining ground as a tool for recording the actions of hiPSC-CMs. GEVIs are both non-invasive and non-labor intensive — and ArcLight is among the most advanced of the lot.

But ArcLight has its own issues, says Deborah Lieu, Ph.D., of the UC Davis Institute for Regenerative Cures and Division of Cardiovascular Medicine, who served as lead investigator on the new study. Mainly, they have to do with how ArcLight was being incorporated into the hiPSCs.

"Prior to our study, in all cases the ArcLight expression depended on lentiviral transduction," she explained. "While lentivirus provides ease of delivery and high expression, it contains transgenes that can randomly integrate into the host genome. This, in turn, can lead to unpredictable consequences such as gene disruption, alteration of global and local gene expression, loss or silencing of reporter genes after differentiation and more."

The trick then, was to find a way to insert ArcLight into the hiPSC-CM line without using a lentivirus. To do so, the Lieu team turned to CRISPR/Cas9 gene editing. CRISPR/Cas9 was already being used successfully to integrate genes and fluorescent reporters into iPSCs. Additionally, tools are available from shared resources and commercial sources that make gene editing relatively cheap, easy and fast. So the Lieu team hypothesized that it could be used to insert ArcLight into the hiPSC-CMs without a negative impact on function.

To begin, they cloned an ArcLight reporter gene into an AAVS1 destination vector. (AAVS1 is considered a "safe harbor site" in gene editing, which means it is an ideal spot in the genome to add a construct without harm.) Next, they used CRISPR/Cas9 gene editing to insert the ArcLight gene into the hiPSCs. They then confirmed their hypotheses that knock-in of ArcLight in the AAVS1 site did not affect the hiPSCs' proliferation and pluripotency.

Not only did this prove true, but they also found that the CRISPR-generated ArcLight reporter line demonstrated better optical performances in terms of photo bleach and phototoxicity than FluoVolt dye.

"Additionally, we learned that ArcLight expression in the hiPSC line persisted following myocardial differentiation. This long-term expression allowed repeated assessment of action potentials in hiPSC-derived cardiomyocytes," said Yao-Hui Sun, Ph.D., the first author and developer of the cell line. "Upon further improvement, this line could be a useful tool for studying cardiac development and drug testing. It might also prove to be useful in characterization of other cell lineages, such as neurons after neuronal differentiation."

"Development of the ArcLight IPSC line will allow straightforward assessment of the function of cardiomyocyte progeny derived from it," said Dr. Jan Nolta, Editor-in-Chief of STEM CELLS. "This line will be an important tool for drug screening and for developing future therapeutics."

The full article, "Human induced pluripotent stem cell line with genetically encoded fluorescent voltage indicator generated via CRISPR for action potential assessment post-cardiogenesis," can be accessed at https://stemcellsjournals.onlinelibrary.wiley.com/doi/abs/10.1002/stem.3085.

About the Journal: STEM CELLS, a peer reviewed journal published monthly, provides a forum for prompt publication of original investigative papers and concise reviews. The journal covers all aspects of stem cells: embryonic stem cells/induced pluripotent stem cells; tissue-specific stem cells; cancer stem cells; the stem cell niche; stem cell epigenetics, genomics and proteomics; and translational and clinical research. STEM CELLS is co-published by AlphaMed Press and Wiley.

About AlphaMed Press: Established in 1983, AlphaMed Press with offices in Durham, NC, San Francisco, CA, and Belfast, Northern Ireland, publishes three internationally renowned peer-reviewed journals with globally recognized editorial boards dedicated to advancing knowledge and education in their focused disciplines. STEM CELLS® (http://www.StemCells.com) is the world's first journal devoted to this fast paced field of research. THE ONCOLOGIST® (http://www.TheOncologist.com) is devoted to community and hospital-based oncologists and physicians entrusted with cancer patient care. STEM CELLS TRANSLATIONAL MEDICINE® (http://www.StemCellsTM.com) is dedicated to significantly advancing the clinical utilization of stem cell molecular and cellular biology. By bridging stem cell research and clinical trials, SCTM will help move applications of these critical investigations closer to accepted best practices.

About Wiley: Wiley, a global company, helps people and organizations develop the skills and knowledge they need to succeed. Our online scientific, technical, medical and scholarly journals, combined with our digital learning, assessment and certification solutions, help universities, learned societies, businesses, governments and individuals increase the academic and professional impact of their work. For more than 200 years, we have delivered consistent performance to our stakeholders. The company's website can be accessed at http://www.wiley.com.

SOURCE STEM CELLS

Related Links

http://www.StemCells.com

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