NEW YORK (GenomeWeb News) – By profiling microRNAs in a range of mouse blood system related cells, Canadian researchers have identified telltale miRNA expression patterns delineating various blood cell lineages at different stages of differentiation.
The British Columbia team relied on real-time quantitative PCR and microfluidics to systematically look at expression patterns for hundreds of miRNAs in dozens of cell varieties isolated from adult mouse hematopoietic tissues. The research, scheduled to appear online this week in the Proceedings of the National Academy of Sciences, is helping to clarify the relationships between these hematopoietic cells, they explained, and highlights the miRNA shifts that occur during differentiation.
"Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage," corresponding author Carl Hansen, a physics and astronomy researcher with the University of British Columbia's Centre for High-Throughput Biology, and his co-authors wrote.
"[M]iRNA expression is very tightly regulated within highly purified populations," they added, "underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity."
During hematopoiesis, the team explained, pluripotent stem cells in the bone marrow become differentiated and specialized to produce a range of different blood related cell types — from white blood cells contributing to immune system function to red blood cells and platelets. In humans, this process occurs every day and is controlled by many different cellular and molecular mechanisms, including miRNAs.
In an effort to understand the miRNA repertoires present during various stages of hematopoiesis and in different cell populations, the researchers developed an approach that relies on multiplex stem-loop RT-qPCR combined with high-throughput qPCR using Fluidigm microfluidic arrays.
After tweaking their protocol in modified mouse bone marrow cells, the team assessed expression patterns for 288 miRNAs in 27 hematopoietic cell populations, such as bone marrow, peripheral blood, and spleen cells, from healthy adult mice.
Using this approach, they detected 187 of the miRNAs in one or more cell population tested. When they assessed the overall miRNA expression profiles in the various cells, the researchers found that these expression patterns generally clustered based on the cell population tested, with more differentiated cells showing distinct miRNA patterns compared to those in stem or progenitor cells.
"The dramatic remodeling of miRNA expression upon commitment to a specific lineage suggests that there is a stem cell signature of miRNA expression, as has been shown for [messenger RNA] expression," they wrote.
In addition, the authors explained, miRNA expression profiles within differentiated blood cells tended to be more similar for cell types with similar functions. The miRNA profiles resembled one another in lymphoid cells, for example, but were distinct from those detected in erythroid cells.
Even so, researchers didn't detect any miRNAs whose expression was restricted to a single cell population. Rather, their findings suggest specific miRNA sets seem to get up- and down-regulated as blood cells differentiate, leading to a host of signaling changes within these cells.
Together, the results support the notion that miRNA profiles vary depending on a cell's level of differentiation as well as its lineage.
And because the expression of these miRNAs seems to be quite stable and tightly regulated, those involved in the study argued that miRNA profiling might serve as a useful tool for identifying, purifying, and distinguishing between cell types.
"The tighter regulation of miRNA is understandable given their global role in post-transcriptional regulation, increased stability relative to mRNA, and high copy numbers," they concluded. "Single-cell miRNA profiling of sorted populations may therefore prove to be a novel and powerful approach for evaluating the purity of populations and for identifying new sub-classes of cells that are not resolved by known surface markers, including cells present in malignant populations or other disease states."