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Researchers Use RNAi to Examine Role Genes Play in Stem Cell Differentiation

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Researchers are working to harness the potential of RNA interference for a wide variety of applications, ranging from simple target validation to combating bioterror. Now, with a grant from the National Center for Research Resources, Brown Medical School assistant professor Bharat Ramratnam — who has been researching the use of RNAi to fight HIV-1 for some time — is adding to this list the use of the technology to understand the genetic basis of stem cell differentiation.

“As a stem cell marches along, people don’t really know what signals make it [differentiate],” Ramratnam told RNAi News. “We thought that the best way to address this was to use RNAi … to systematically go after certain eukaryotic transcription factors that have been studied previously, but not in adult systems [such as] adult rodents.”

Ramratnam’s effort is being funded as part of a broad, five-year program designed to bring together mentoring investigators at established institutions and young scientists to work on advancing stem cell research. The NCRR has earmarked roughly $13.3 million for the project, with about $3.1 million having been already provided this year.

The initial stage of Ramratnam’s work will involve silencing PU1 — a transcription factor that controls many genes important in myeloid cells — in primary murine hematopoietic stem cells by delivering shRNA with viral vectors. Ramratnam and colleagues at Roger Williams Hospital will then assess the results of the knockdown in vitro, with the goal of finding out how PU1 disruption impacts myeloid cell differentiation, gene expression, and bone marrow repopulation in vivo.

“Why RNAi fits in this model, is that if you have a PU1 knockout, its embryonic lethal,” he said. “There’s really no way of studying its genetic disruption in the adult animal, so you don’t have a complete picture.” But by silencing PU1 in stem cells and then introducing them into an adult animal, Ramratnam said he hopes to gain a better idea of what role PU1 plays in the differentiation of stem cells into myeloid cells.

After about a month’s work that he characterized as an empiric process, Ramratnam said that he and his colleagues have come up with an shRNA that knocks down PU1. They have also transferred the shRNA into adeno-associated vectors and just begun transfecting them into murine stem cells in vitro. He said that he expects to begin seeing results before the end of the year, after which he plans to begin experimenting in animal models.

“This is a proof-of-principle experiment” to show that in vivo data can be gathered using this approach, Ramratnam said. While they are starting out looking at PU1, largely because “it seems as valid [a target] as any other,” he and his colleagues are also planning to examine other transcription factors, such as GATA-1, as well as interactions between multiple transcription factors that affect myeloid cells.

However, “we haven’t really thought about that [just yet],” he said. “We’re just focused on making sure this works.”

While this project is not expected to result in the development of a specific therapeutic, Ramratnam said, “it may help us understand how we can engineer a stem cell to go to an organ we want [or] change into a cell we want it to change into — how we can manipulate these stem cells.

“For example, in tissue injury, if we ever get to the point we can reconstitute an organ with stem cells, when we introduce the stem cells, we have to be sure they’re going to do what we want them to do,” he said. “So by knocking down factors that impact [the stem cells’] long march ahead, or by knocking down homing molecules that tell the stem cell where to go, we may be able to further that goal.”

“RNAi is a tool that will just be applied everywhere,” he added. While people mostly discuss the technology in terms of the immediate translation of this into a therapeutic approach, “in the long run, it’s going to be a pathogenesis approach.

“In model systems that are very well characterized, where you just want to analyze what is perturbing one, two, or three different proteins or RNAs, this is the way to go.”

—DM

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