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A simple way for stem cells to become heart cells
The process by which embryonic stem cells develop into heart cells is complex. It involves precisely timed activation of several molecular pathways, and at least 200 genes. Now, Salk Institute scientists have found a simpler way to go from stem cells to heart cells by turning off a single gene.
The work, which appears in Genes & Development on December 21, 2017, offers scientists a streamlined method to arrive at functioning heart cells (cardiomyocytes) for both research and regenerative therapies.
"This discovery is really exciting because it means we can potentially create a reliable protocol for taking normal cells and moving them efficiently from stem cells to heart cells. Researchers and commercial companies want to easily generate cardiomyocytes to study their capacity for repair in heart attacks and disease — this brings us one step closer to being able to do that."
In 2015, Jones' lab, which studies proteins that manage cell growth and development, discovered two different cellular processes cooperate enabling embryonic stem cells (ESCs) to develop into specific cell types like pancreas, liver and heart. Her team found (1) the Wnt pathway loads up cellular machinery and begins copying and activating genes, and then (2) the Activin pathway ramps up that activity. Together, the two pathways (named for their key proteins) direct stem cells into an intermediate stage from which ESCs further progress into cells of specific organs. By exposing the cells to a signaling molecule at two different timepoints, the team could trigger first Wnt then Activin and end up with specialized cells.
In this process, the team also discovered a third pathway — governed by a protein called YAP — which seemed to put the brakes on the Activin pathway, keeping stem cells from specializing.
Wanting to better understand how, the current work by Jones and first author Conchi Estarás set out to manipulate the YAP gene in various ways to see what would happen. They began by using the molecular scissors known as CRISPR-Cas9 to cut the gene out of the DNA in ESCs,' so they could no longer make the YAP protein. They then exposed them to the signaling molecule to see what, if anything, happened.
To their great surprise, the cells went from the stem cell stage directly to beating heart cells.
"Instead of requiring two steps to achieve specialization, removing YAP cut it to just one step," says Estarás, who is a Salk research associate. "That would mean a huge savings for industry in terms of reagent materials and expense."
Intriguingly, further analysis revealed that the same genes were being turned on as would be activated via the normal Wnt-Activin stem-cell specialization process.
"This revealed to us a hidden, specific cellular lineage directly to beating cardiomyocytes," says Jones. "It's both fascinating and medically and commercially useful to find genes that are differently regulated still lead to the same result."
Because removing a gene entirely can have unintended effects, the team next wants to test whether they can turn off YAP using small commercially available inhibitor molecules, and still derive functioning cardiac cells from stem cells.
Activin/SMAD signaling in human embryonic stem cells (hESCs) ensures NANOG expression and stem cell pluripotency. In the presence of Wnt ligand, the Activin/SMAD transcription network switches to cooperate with Wnt/?-catenin and induce mesendodermal (ME) differentiation genes. We show here that the Hippo effector YAP binds to the WNT3 gene enhancer and prevents the gene from being induced by Activin in proliferating hESCs. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data show that YAP impairs SMAD recruitment and the accumulation of P-TEFb-associated RNA polymerase II (RNAPII) C-terminal domain (CTD)-Ser7 phosphorylation at the WNT3 gene. CRISPR/CAS9 knockout of YAP in hESCs enables Activin to induce Wnt3 expression and stabilize ?-catenin, which then synergizes with Activin-induced SMADs to activate a subset of ME genes that is required to form cardiac mesoderm. Interestingly, exposure of YAP-/- hESCs to Activin induces cardiac mesoderm markers (BAF60c and HAND1) without activating Wnt-dependent cardiac inhibitor genes (CDX2 and MSX1). Moreover, canonical Wnt target genes are up-regulated only modestly, if at all, under these conditions. Consequently, YAP-null hESCs exposed to Activin differentiate precisely into beating cardiomyocytes without further treatment. We conclude that YAP maintains hESC pluripotency by preventing WNT3 expression in response to Activin, thereby blocking a direct route to embryonic cardiac mesoderm formation.
Authors: Conchi Estarás, Hui-Ting Hsu, Ling Huang and Katherine A. Jones
The work was funded by the California Institute for Regenerative Medicine and the National Cancer Institute (NIH)
About the Salk Institute for Biological Studies:
Every cure has a starting point. The Salk Institute embodies Jonas Salk's mission to dare to make dreams into reality. Its internationally renowned and award-winning scientists explore the very foundations of life, seeking new understandings in neuroscience, genetics, immunology, plant biology and more. The Institute is an independent nonprofit organization and architectural landmark: small by choice, intimate by nature and fearless in the face of any challenge. Be it cancer or Alzheimer's, aging or diabetes, Salk is where cures begin. Learn more at: salk.edu.
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This cartoon illustrates how Yap (DOG) inhibits expression of Wnt (Girl in Red Bathing Suit), preventing an intermediate complex (Boy and Girl sitting on Beach Ball) from binding to RNA (Girl in Yellow Suit) and signaling various types of cell machinery (Girl Diving off platform, and Boy seated on platform) to turn on the cardiomyocyte genes (Boy Swimming in ocean).
Image credit: Salk Institute.