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Scientists know that cellular reprogramming can reverse the process of cellular aging that leads to a decline in the activities and functions of mesenchymal stem/stromal cells (MSCs), but the underlying mechanisms haven’t been clear. Newly reported research by scientists at the University of Wisconsin-Madison has identified a key role for a protein known as GATA6, in this reversal process. Their results, published in Stem Cells, provide new insights into MSC aging and associated diseases, which could help lead to the development of pharmacological strategies to reduce or reverse the aging process.

“While agreeing with previous findings in MSC rejuvenation by cellular reprogramming, our study goes further to provide insight into how reprogrammed MSCs are regulated molecularly to ameliorate the cellular hallmarks of aging,” explained lead investigator, Wan-Ju Li, PhD, a faculty member in the Department of Orthopedics and Rehabilitation and the Department of Biomedical Engineering. “We believe our findings will help improve the understanding of MSC aging and its significance in regenerative medicine.”

Li and colleagues report their findings in a paper titled, “GATA6 regulates aging of human mesenchymal stem/stromal cells.”

Aging leads to a decline in the activities and functions of tissue-derived mesenchymal stem cells (MSCs), and this is considered to be a major challenge for the future development of MSC therapies and tissue engineering, the authors wrote. While scientists can use a range of model organisms—including yeast, Caenorhabditis elegans, Drosophila melanogaster— to investigate mechanisms of aging, it’s now possible to harness cellular reprogramming as a tool for manipulating the cellular aging process in human cells, and provide a better understanding of underlying mechanisms.

“Previous studies have demonstrated that direct cellular reprogramming with the use of Yamanaka factors rejuvenates bone marrow-derived MSCs with evidence indicating changes in cellular characteristics, DNA methylation profiles, or aging signature genes between reprogrammed MSCs derived from iPSCs (iPSCMSCs) and nonreprogrammed parental MSCs,” the team stated. “ … cellular reprogramming has been harnessed as an effective approach to ameliorate aging hallmarks in a variety of cultured human cells and restore the regenerative capability to aged muscle stem cells implanted in animals.” The Yamanaka transcription factors are four reprogramming factors that are used to derive the iPSCs.

Li and colleagues used cellular reprogramming to establish a genetically identical young and old cell model. They began by isolating MSCs from human synovial fluid (SF-MSCs), and reprogrammed them into induced pluripotent stem cells (iPSCs). Then they reverted these iPSCs back to MSCs, in effect rejuvenating the MSCs. “When we compared the reprogrammed MSCs to the non-rejuvenated parental MSCs, we found that aging-related activities were greatly reduced in reprogrammed MSCs compared to those in their parental lines. This indicates a reversal of cell aging,” Li said.

When mesenchymal stem/stromal cells (MSCs) age, the transcription factor GATA6 is increasingly produced in the cell to induce aging response. By transcription factor-based cellular reprogramming, aged MSCs are rejuvenated with a reduction in GATA6 effects on cellular aging. [AlphaMed Press]
“Using the parental and reprogrammed MSCs as control nonrejuvenated and rejuvenated cells, respectively, for comparative analysis, we found that aging-related activities were greatly reduced in reprogrammed MSCs compared with those in their parental lines, indicating reversal of cell aging,” the team wrote in their paper.

 

The scientists next conducted an analysis of the cells to determine if there were any changes in global gene expression resulting from the reprogramming. They found that the expression of GATA6, a protein that plays an important role in gut, lung, and heart development, was repressed in the reprogrammed cells compared with the control cells. This repression led to increased activity of a protein called sonic hedgehog (SHH) that is essential to embryonic development, as well as the expression level of another protein, FOXP1, which is necessary for proper development of the brain, heart, and lung.

“Mechanistically, we demonstrated that, compared with control cells, the expression of GATA binding protein 6 (GATA6) in reprogrammed cells was attenuated, resulting in an increase in the activity of sonic hedgehog signaling and the expression level of downstream forkhead box P1 (FOXP1), in turn ameliorating cellular hallmarks of aging,” they wrote. “Thus, we identified the GATA6/SHH/FOXP1 pathway as a key mechanism that regulates MSC aging and rejuvenation,” Li claimed.

To determine which of the Yamanaka transcription factors were involved in repressing GATA6 in the iPSCs, the team analyzed GATA6 expression in response to the knockdown of each factor. The results indicated that only OCT4 and KLF4 were able to regulate GATA6 activity, a finding that is consistent with that of several previous studies. “To determine which of Yamanaka factors is involved in repressing GATA6 in iPSCs, we analyzed GATA6 expression in response to knockdown of the factors and have found that only OCT4 and KLF4 are able to regulate GATA6 activity,” they wrote.

“Overall, we were able to demonstrate that SF-MSCs undergo substantial changes in properties and functions as a result of cellular reprogramming,” Li said. “These changes in iPSC-MSCs collectively indicate amelioration of cell aging. Most significantly, we were able to identify the GATA6/SHH/FOXP1 signaling pathway as an underlying mechanism that controls cell aging-related activities.”

The authors concluded, “Our results, while agreeing with previous findings in MSC rejuvenation by cellular reprogramming, provide insight into how reprogrammed MSCs are regulated molecularly to ameliorate cellular hallmarks of aging … These findings, critical to enhancing the knowledge of MSC senescence and diseases associated with MSC aging and to providing insight into developing pharmacological strategies to ameliorate cellular aging, are considered significant and timely for regenerative medicine.”

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