“Not just the FACS, ma’am, not just the FACS.” These words could have come from investigators who have applied a new technique to assess gene expression in stem cells. FACS, or fluorescence-activated cell sorting, is used to remove cells from their native environment and purify them for analysis. Another, newer technique, however, may better reflect stem cells’ in vivo state.
The new technique, which labels newly transcribed RNA in vivo, has been applied by scientists based at Stanford University School of Medicine. According to these scientists, the in vivo approach reveals transcriptional nuances that cannot be captured by FACS, whether this isolation procedure is combined with ordinary culture techniques, or specialized techniques that temporarily arrest transcription.
The Stanford team, led by Thomas Rando, M.D., Ph.D., professor of neurology and neurological sciences, focused on muscle stem cells (MuSCs) in mice. When snug in the body, MuSCs tend to remain quiescent, actively maintaining a state that looks dormant, but isn’t. When extracted from the body, MuSCs tend to become slightly roused, even if they remain quiescent.
“Historically, we've thought of quiescence as an 'everything off,' or dormant, state,” said Dr. Rando. “But our work has shown that the reality is quite different. Not only have we been missing transcripts that are present in vivo, but we are also puzzled as to why so many transcripts that are made in vivo are not made into proteins.
“It's possible that this is one way the cells stay ready to undergo a rapid transformation, either by blocking degradation of RNA or proteins or by swiftly initiating translation of already existing RNA transcripts.”
To advance the exploration of these possibilities, Dr. Rando and colleagues published an article (“Transcriptional Profiling of Quiescent Muscle Stem Cells In Vivo”) that appeared November 14 in the journal Cell Reports. The article describes how the researchers applied a technique for labeling newly transcribed RNA in vivo in a cell-specific manner.
The technique involves engineering cells to express the Toxoplasma gondii uracil phosphoribosyltransferase (UPRT). Thus modified, the cells can convert 4-thiouracil (4tU) into 4-thiouridine mono-phosphate, which is then be incorporated into the RNA that is being transcribed.
In the current study, the researchers generated mice in which UPRT is expressed specifically in MuSCs, thereby allowing RNA to be labeled in vivo.
“Here, we analyze the transcriptome of MuSCs in vivo utilizing MuSC-specific labeling of RNA,” wrote the authors of the Cell Reports article. “Notably, labeling transcripts during the isolation procedure revealed very active transcription of specific subsets of genes.”
The researchers found that isolated cells make large numbers of RNA molecules known to be involved in cellular stress and in cellular proliferation. Conversely, stem cells in the body make more RNAs involved in maintaining the quiescent state, in which they exist until called upon to make new muscle fibers.
The researchers additionally found that the process of isolating whole muscle stem cells for study caused some important RNA molecules to be degraded, rendering them undetectable in previous studies. These findings further support the notion that this quiescent state is not one of dormancy, but one of active regulation and controls—controls that are no longer needed once the cells are awakened to begin the process of tissue repair.
In the Cell Reports article, the authors noted that when the transcription inhibitor α-amanitin is used, the ex vivo transcriptome remains largely reflective of the in vivo transcriptome. The key word here being “largely.” “[Our] labeling approach, the authors added, “yielded information on transcript dynamics in MuSCs in vivo in ways that steady-state transcriptome analysis cannot.”
“The cells in the animal clearly differ from those that are removed for study,” insisted Dr. Rando. “It's likely that some of these notable differences will skew our view of what the quiescent state entails for many types of adult stem cells. We and other researchers will need to rethink about how to profile stem cells in a way that accurately reflects their in vivo state.”
Dr. Rando and his colleagues expect that the new RNA labeling technique will be used by many other researchers studying adult stem cells.
“It's so important to know what we are and are not modeling about the state of these cells in vivo,” emphasized Dr. Rando. “Are we modeling it correctly when we look at stem cells isolated by FACS? This study will have a big impact on how researchers in the field think about understanding the characteristics of stem cells as they exist in their native state in the tissue.”