Evidence for Diversity in Transcriptional Profiles of Single Hematopoietic Stem Cells

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Abstract
Hematopoietic stem cells replenish all the cells of the blood throughout the lifetime of an animal. Although thousands of stem cells reside in the bone marrow, only a few contribute to blood production at any given time. Nothing is known about the differences between individual stem cells that dictate their particular state of activation readiness. To examine such differences between individual stem cells, we determined the global gene expression profile of 12 single stem cells using microarrays. We showed that at least half of the genetic expression variability between 12 single cells profiled was due to biological variation in 44% of the genes analyzed. We also identified specific genes with high biological variance that are candidates for influencing the state of readiness of individual hematopoietic stem cells, and confirmed the variability of a subset of these genes using single-cell real-time PCR. Because apparent variation of some genes is likely due to technical factors, we estimated the degree of biological versus technical variation for each gene using identical RNA samples containing an RNA amount equivalent to that of single cells. This enabled us to identify a large cohort of genes with low technical variability whose expression can be reliably measured on the arrays at the single-cell level. These data have established that gene expression of individual stem cells varies widely, despite extremely high phenotypic homogeneity. Some of this variation is in key regulators of stem cell activity, which could account for the differential responses of particular stem cells to exogenous stimuli. The capacity to accurately interrogate individual cells for global gene expression will facilitate a systems approach to biological processes at a single-cell level. The hematopoietic stem cell (HSC) has the remarkable property of being able to generate more stem cells or cells that are committed to undergo differentiation into specific blood lineages. Currently, very little is known about the specific mechanisms that underlie self-renewal or lineage commitment. Although it is possible that some of these mechanisms are influenced by the specific environment in which the HSC dwells, the ultimate fate decision has to occur at the single HSC level. The authors have developed a method that amplifies the messages from the majority of genes that are active in a single stem cell and combines it with large-scale genetic expression analysis through the use of nucleic acid microarrays. A significant fraction of these genes are found to be highly variable in an apparently very homogeneous stem cell population, which could be the substrate for differences in behavior of individual stem cells. Understanding the genetic expression events at the single-cell level would grant the ability to expand HSCs or to direct their differentiation into specific populations, both important from a therapeutic point of view. Furthermore, the same techniques can be applied to other stem cell systems to investigate their physiology.