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.
Highlights
Interest in adult stem cells has intensified since 2002, due to renewed hope for their application to regenerative medicine [1,2,3,4,5,6,7,8]
Our group has recently demonstrated that side population (SP) cells are phenotypically LinÀ/lowSca-1þc-kitþThy1.1low CD34neg/lowFlk2neg and are, in essence, the same hematopoietic stem cell (HSC) population isolated by other groups
We found that several genes previously described in association with HSC populations, such as Sca-1, LIM domain only 2 (Lmo2), Ctla-2a, and thrombopoietin receptor (TPO-R), were found to be present by our method in each of the single stem cells studied
Summary
Interest in adult stem cells has intensified since 2002, due to renewed hope for their application to regenerative medicine [1,2,3,4,5,6,7,8]. An adult mouse harbors hundreds of HSCs, only between one and ten are thought to be active in contributing to blood production at any time [9,10]. Nothing is known about the mechanisms that favor activation of one stem cell over another. Apart from micro-environmental factors, there are individual differences in the ability of particular stem cells to respond, based on a constellation of response genes they express at a given time. Several efforts have been made to study the transcriptional profile of HSC at the population level [11,12,13,14,15,16,17], the ability to investigate gene expression in stem (and other) cells at the single-cell level would be a powerful tool to understand their biology
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