Generation of an hESC line deficient in HLA-I/II and MICA/B (A) Representation of the Cas9 editing strategy. Wild-type H1 hESCs were transfected with a Cas9 construct and up to three gRNAs at a time. The gRNAs were for β2M, TAP1, CD74, CIITA, MICA and MICB. Individual colonies were selected and sequenced to identify those with frameshift mutations in target genes. Colonies with mutations were clonally sorted and resequenced. The resulting cells were then iteratively targeted with gRNAs to generate a line in which all alleles carried frameshift mutations. (B) Representative flow cytometric histograms of HLA-ABC (HLA-I) expression on H1 and HM-KO hESCs treated with PBS or IFNγ. (C) Representative histograms of HLA-DR (HLA-II) expression of DC-like cultures differentiated from H1 or HLA-I/II-KO hESCs. Credit: Stem Cell Reports (2024). DOI: 10.1016/j.stemcr.2023.12.003
One of the biggest obstacles to regenerative medicine is immunological rejection of the recipient, a problem that University of Arizona Health Sciences researchers are close to solving after genetically modifying pluripotent stem cells to escape immune recognition.
Their study entitled “Engineering human pluripotent stem cell lines to escape xenogenic barriers in transplantation” is published in the journal Stem Cell Reports.
Pluripotent stem cells can develop into any type of cell in the body. The findings provide a viable avenue for pluripotent stem cell-based therapies to restore tissue lost in diseases such as type 1 diabetes or macular degeneration.
“There has been a lot of excitement for decades around the field of pluripotent stem cells and regenerative medicine,” said principal investigator Deepta Bhattacharya, Ph.D., professor in the Department of Immunobiology at UArizona College of Medicine in Tucson.
“What we have learned from organ transplant experiences is that you need to have compatible donors, but the person receiving the transplant often still requires lifelong immunosuppression, which means that it is more susceptible to infections and cancer. We’ve been trying to figure out what you need to do with these stem cells to keep them from being rejected, and it looks like we have a possible solution.
To test their hypothesis, Bhattacharya and the research team used CRISPR-Cas9 technology, “genetic scissors” that allow scientists to make precise mutations in the genome at extremely specific locations.
Using human pluripotent stem cells, the team located specific genes they believed to be involved in immune rejection and deleted them. Previous research on pluripotent stem cells and immune rejection had looked at different parts of the immune system in isolation. Bhattacharya and his colleagues at the New York Stem Cell Foundation Research Institute, St. Jude Children’s Research Hospital, and Washington University School of Medicine chose to test their genetically engineered stem cells in a complete, functioning immune system.
“The immune system is really complicated and there are all kinds of ways to recognize and reject things,” said Bhattacharya, a member of the UArizona Cancer Center and BIO5 Institute who also serves in the Health Sciences Center at the UArizona for molecular and immunological therapies. advisory board.
“Transplantation between species, across the xenogenic barrier, is difficult and sets a very high bar for transplantation. We decided that if we could overcome this barrier, then we could begin to have confidence in our ability to overcome this which should be a simpler approach to cross-species transplantation. human barrier, and so that’s essentially what we did.
The research team tested the modified stem cells by placing them in mice with normal, fully functioning immune systems. The results were promising: the genetically modified pluripotent stem cells were integrated and persisted without being rejected.
“This has been the Holy Grail for a while. You might actually have a chance of being able to perform pluripotent stem cell transplants without immunosuppression of the person receiving them. That would be an important advance, both clinically and from a point of view. from a clinical point of view. just a scale point of view,” Bhattacharya said. “You wouldn’t need to come up with individualized therapies for each person: you could start with one type of pluripotent stem cell, change it to the cell type you want, and then give it to almost everyone.”
Next steps, Bhattacharya said, include testing genetically engineered pluripotent stem cells in specific disease models. He is already working with collaborators at the New York Stem Cell Foundation and the Juvenile Diabetes Research Foundation to test the technology in animal models for type 1 diabetes.
“We had to defeat the immune system first. The next steps are how do we use these cells?” Bhattacharya said. “We set the bar pretty high for our study and the fact that we were successful gives us some confidence that this can really work.”
More information:
Hannah A. Pizzato et al, Engineering human pluripotent stem cell lines to escape xenogenic barriers of transplantation, Stem Cell Reports (2024). DOI: 10.1016/j.stemcr.2023.12.003
Provided by the University of Arizona
Quote: Study shows that genetically modified pluripotent stem cells can escape immunological rejection after transplantation (January 11, 2024) retrieved January 11, 2024 from
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