Evidence for diversity in transcriptional profiles of single hematopoietic stem cells.
Evidence for diversity in transcriptional profiles of single hematopoietic stem cells.
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DOI：
10.1371/journal.pgen.0020159
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发表时间：
2006-09-29
期刊：
影响因子：
4.5
通讯作者：
Goodell MA
中科院分区：
文献类型：
--
作者：
Ramos CA;Bowman TA;Boles NC;Merchant AA;Zheng Y;Parra I;Fuqua SA;Shaw CA;Goodell MA
造血干细胞在动物的一生中补充血液中的所有细胞。虽然成千上万的干细胞存在于骨髓中，但在任何特定时间只有少数干细胞有助于造血。目前还不清楚个体干细胞之间的差异，这些差异决定了它们特定的激活准备状态。为了研究个体干细胞之间的这种差异，我们使用微阵列确定了12个单个干细胞的整体基因表达谱。我们发现，12个单细胞之间至少有一半的基因表达变异是由于44%的基因分析的生物学变异。我们还确定了具有高生物学差异的特定基因，这些基因是影响个体造血干细胞准备状态的候选基因，并使用单细胞实时PCR证实了这些基因的子集的变异性。由于某些基因的明显变异可能是由于技术因素造成的，因此我们使用含有相当于单细胞RNA量的相同RNA样品来估计每个基因的生物学与技术变异的程度。这使我们能够识别出大量具有低技术变异性的基因，这些基因的表达可以在单细胞水平上在阵列上可靠地测量。这些数据已经确定，尽管表型同质性极高，但个体干细胞的基因表达差异很大。其中一些变化是在干细胞活性的关键调节因子中，这可以解释特定干细胞对外源刺激的差异反应。准确地询问单个细胞的全局基因表达的能力将促进在单细胞水平上的生物过程的系统方法。造血干细胞（HSC）具有能够产生更多干细胞或致力于经历分化成特定血液谱系的细胞的显著特性。目前，人们对自我更新或血统承诺的具体机制知之甚少。尽管这些机制中的一些可能受到HSC所处的特定环境的影响，但最终的命运决定必须发生在单个HSC水平上。作者开发了一种方法，可以放大单个干细胞中活跃的大多数基因的信息，并通过使用核酸微阵列将其与大规模遗传表达分析相结合。这些基因中的一个重要部分被发现是高度可变的，在一个明显非常同质的干细胞群体，这可能是基板的行为差异的个别干细胞。了解单细胞水平的基因表达事件将赋予扩增HSC或将其分化为特定群体的能力，从治疗的角度来看，这两者都很重要。此外，同样的技术可以应用于其他干细胞系统，以研究它们的生理学。
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.
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DOI：
10.1073/pnas.0538056100
发表时间：
2003-03-04
影响因子：
11.1
作者：
Johnson, BJ;Costelloe, EO;Kelso, A
通讯作者：
Kelso, A
影响因子：
16
作者：
Cho, RJ;Campbell, MJ;Davis, RW
通讯作者：
Davis, RW
影响因子：
11.8
作者：
Chiang, MK;Melton, DA
通讯作者：
Melton, DA
影响因子：
56.9
作者：
Brazelton, TR;Rossi, FMV;Blau, HM
通讯作者：
Blau, HM
影响因子：
20.3
作者：
Camargo, FD;Chambers, SM;Goodell, MA
通讯作者：
Goodell, MA